Inhibition of HIV-1 Replication by Disruption of the Processing of the Viral Capsid-Spacer Peptide 1 Protein

ABSTRACT

Inhibition of HIV-1 replication by disrupting the processing of the viral Gag capsid (CA) protein (p24) from the CA-spacer peptide 1 (SP1) protein precursor (p25) is disclosed. Amino acid sequences containing a mutation in the Gag p25 protein, with the mutation resulting in a decrease in the inhibition of processing of p25 to p24 by dimethylsuccinyl betulinic acid or dimethylsuccinyl betulin, polynucleotides encoding such mutated sequences and antibodies that selectively bind such mutated sequences are also included. Methods of inhibiting, inhibitory compounds and methods of discovering inhibitory compounds that target proteolytic processing of the HIV Gag protein are included. In one embodiment, such compounds inhibit the interaction of the HIV protease enzyme with Gag by binding to Gag rather than to the protease enzyme. In another embodiment, viruses or recombinant proteins that contain mutations in the region of the Gag proteolytic cleavage site can be used in screening assays to identify compounds that target proteolytic processing.

RELATED U.S. APPLICATION DATA

This application is a divisional of U.S. application Ser. No.10/851,637, filed May 24, 2004, which is a continuation-in-part of U.S.application Ser. No. 10/706,528, filed Jan. 29, 2004, which claims thebenefit of U.S. Provisional Application Nos. 60/496,660, filed Aug. 21,2003, and 60/443,180, filed Jan. 29, 2003, all of which are entirelyincorporated by reference herein.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of Grant No.2R44AI051047-02 awarded by NIH/NIAID.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention includes methods of inhibiting, inhibitors and methods ofdiscovery of inhibitors of HIV infection.

2. Background

Human Immunodeficiency Virus (HIV) is a member of the lentiviruses, asubfamily of retroviruses. The viral genome contains many regulatoryelements which allow the virus to control its rate of replication inboth resting and dividing cells. Most importantly, HIV infects andinvades cells of the immune system; it breaks down the body's immunesystem and renders the patient susceptible to opportunistic infectionsand neoplasms. The immune defect appears to be progressive andirreversible, with a high mortality rate that approaches 100% overseveral years.

HIV-1 is trophic and cytopathic for T4 lymphocytes, cells of the immunesystem which express the cell surface differentiation antigen CD4, alsoknown as OKT4, T4 and leu3. The viral tropism is due to the interactionsbetween the viral envelope glycoprotein, gp120, and the cell-surface CD4molecules (Dalgleish et al., Nature 312:763-767 (1984)). Theseinteractions not only mediate the infection of susceptible cells by HIV,but are also responsible for the virus-induced fusion of infected anduninfected T cells. This cell fusion results in the formation of giantmultinucleated syncytia, cell death, and progressive depletion of CD4cells in HIV-infected patients. These events result in HIV-inducedimmunosuppression and its subsequent sequelae, opportunistic infectionsand neoplasms.

In addition to CD4⁺ T cells, the host range of HIV includes cells of themononuclear phagocytic lineage (Dalgleish et al., supra), includingblood monocytes, tissue macrophages, Langerhans cells of the skin anddendritic reticulum cells within lymph nodes. HIV is also neurotropic,capable of infecting monocytes and macrophages in the central nervoussystem causing severe neurologic damage. Macrophage and monocytes aremajor reservoirs of HIV. They can interact and fuse with CD4-bearing Tcells, causing T cell depletion and thus contributing to thepathogenesis of AIDS.

Considerable progress has been made in the development of drugs forHIV-1 therapy. Therapeutic agents for HIV can include, but not are notlimited to, at least one of AZT, 3TC, ddC, d4T, ddI, tenofovir,abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir,ritonavir, indinavir, nelfinavir, lopinavir, amprenavir, atazanavir andfosamprenavir, or any other antiretroviral drugs or antibodies incombination with each other, or associated with a biologically basedtherapeutic, such as, for example, gp41-derived peptides enfuvirtide(Fuzeon; Timeris-Roche) and T-1249 (Trimeris), or soluble CD4,antibodies to CD4, and conjugates of CD4 or anti-CD4, or as additionallypresented herein. Combinations of these drugs are particularly effectiveand can reduce levels of viral RNA to undetectable levels in the plasmaand slow the development of viral resistance, with resultingimprovements in patient health and life span.

Despite these advances, there are still problems with the currentlyavailable drug regimens. Many of the drugs exhibit severe toxicities,have other side-effects (e.g., fat redistribution) or requirecomplicated dosing schedules that reduce compliance and thereby limitefficacy. Resistant strains of HIV often appear over extended periods oftime even on combination therapy. The high cost of these drugs is also alimitation to their widespread use, especially outside of developedcountries.

There is still a major need for the development of additional drugs tocircumvent these issues. Ideally these would target different stages inthe viral life cycle, adding to the armamentarium for combinationtherapy, and exhibit minimal toxicity, yet have lower manufacturingcosts.

HIV virion assembly takes place at the surface membrane of the infectedcell where the viral Gag polyprotein accumulates, leading to theassembly of immature virions that bud from the cell surface. Within thevirion, Gag is cleaved by the viral proteinase (PR) into the matrix(MA), capsid (CA), nucleocapsid (NC), and C-terminal p6 structuralproteins (Wiegers K. et al., J. Virol. 72:2846-2854 (1998)). Gagprocessing induces a reorganization of the internal virion structure, aprocess termed “maturation.” In mature HIV particles, MA lines the innersurface of the membrane, while CA forms the conical core which encasesthe genomic RNA that is complexed with NC. Cleavage and maturation arenot required for particle formation but are essential for infectivity(Kohl, N. et al, Proc. Natl. Acad. Sci. USA 85:4686-4690, (1998)).

CA and NC as well as NC and p6 are separated on the Gag polyprotein byshort spacer peptides of 14 and 10 amino acids (p2), respectively(spacer peptide 1 (SP1) and SP2, respectively) (Wiegers K. et al., J.Virol. 72:2846-2854 (1998), Pettit, S. C. et al., J. Virol. 68:8017-8027(1994), Liang et al. J. Virol. 76:11729-11737 (2002)). These spacerpeptides are released by PR-mediated cleavages at their N and C terminiduring particle maturation. The individual cleavage sites on the HIV Gagand Gag-Pol polyproteins are processed at different rates and thissequential processing results in Gag intermediates appearing transientlybefore the final products. Such intermediates may be important forvirion morphogenesis or maturation but do not contribute to thestructure of the mature viral particle (Weigers et al. and Pettit, etal., supra). The initial Gag cleavage event occurs at the C terminus ofSP1 and separates an N-terminal MA-CA-SP1 intermediate from a C-terminalNC-SP2-p6 intermediate. Subsequent cleavages separating MA from CA-SP1and NC-SP2 from p6 occur at an approximately 10-fold-lower rate.Cleavage of SP1 from the C terminus of CA is a late event and occurs ata 400-fold-lower rate than cleavage at the SP1-NC site (Weigers et al.and Pettit, et al., supra). The uncleaved CA-SP1 intermediate protein isalternatively termed “p25,” whereas the cleaved CA protein isalternatively termed “p24” and the cleaved SP1 peptide is alternativelytermed “p2”.

Cleavage of SP1 from the C terminus of CA appears to be one of the lastevents in the Gag processing cascade and is required for final capsidcondensation and formation of mature, infectious viral particles.Electron micrographs of mature virions reveal particles having electrondense conical cores. On the other hand, electron microscopy studies ofviral particles defective for CA-SP1 cleavage show particles having aspherical electron-dense ribonucleoprotein core and a crescent-shaped,electron-dense layer located just inside the viral membrane (Weigers etal., supra). Mutations at or near the CA-SP1 cleavage site have beenshown to inhibit Gag processing and disrupt the normal maturationprocess, thereby resulting in the production of non-infectious viralparticles (Weigers et al., supra). Phenotypically, these particlesexhibit a defect in Gag processing (which manifests itself in thepresence of a p25 (CA-SP1) band in Western blot analysis) and theaberrant particle morphology described above which results fromdefective capsid condensation.

Previously, betulinic acid and platanic acid were isolated from Syzigiumclaviflorum and were determined to have anti-HIV activity. Betulinicacid and platanic acid exhibited inhibitory activity against HIV-1replication in H9 lymphocyte cells with EC₅₀ values of 1.4 μM and 6.5μM, respectively, and therapeutic index (T.I.) values of 9.3 and 14,respectively. Hydrogenation of betulinic acid yielded dihydrobetulinicacid, which showed slightly more potent anti-HIV activity with an EC₅₀value of 0.9 and a T.I. value of 14 (Fujioka, T., et al., J. Nat. Prod.57:243-247 (1994)). Esterification of betulinic acid with certainsubstituted acyl groups, such as 3′,3′-dimethylglutaryl and3′,3′-dimethylsuccinyl groups produced derivatives having enhancedactivity (Kashiwada, Y., et al., J. Med. Chem. 39:1016-1017 (1996)).Acylated betulinic acid and dihydrobetulinic acid derivatives that arepotent anti-HIV agents are also described in U.S. Pat. No. 5,679,828.Anti-HIV assays indicated that 3-O-(3′,3′-dimethylsuccinyl)-betulinicacid (DSB) and the dihydrobetulinic acid analog both demonstratedextremely potent anti-HIV activity in acutely infected H9 lymphocyteswith EC₅₀ values of less than 1.7×10⁻⁵ μM, respectively. These compoundsexhibited remarkable T.I. values of more than 970,000 and more than400,000, respectively.

U.S. Pat. No. 5,468,888 discloses 28-amido derivatives of lupanes thatare described as having a cytoprotecting effect for HIV-infected cells.

Japanese Patent Application No. JP 01 143,832 discloses that betulin and3,28-diesters thereof are useful in the anti-cancer field.

U.S. Pat. No. 6,172,110 discloses betulinic acid and dihydrobetulinderivatives which have the following formulae or pharmaceuticallyacceptable salts thereof,

Betulin and Dihydrobetulin Derivatives

wherein R₁ is a C₂-C₂₀ substituted or unsubstituted carboxyacyl, R₂ is aC₂-C₂₀ substituted or unsubstituted carboxyacyl; and R₃ is hydrogen,halogen, amino, optionally substituted mono- or di-alkylamino, or —OR₄,where R₄ is hydrogen, C₁₋₄ alkanoyl, benzoyl, or C₂-C₂₀ substituted orunsubstituted carboxyacyl; wherein the dashed line represents anoptional double bond between C20 and C29.

U.S. Patent Application No. 60/413,451 discloses 3,3-dimethylsuccinylbetulin and is herein incorporated by reference. Zhu, Y-M. et al.,Bioorg. Chem. Lett. 11:3115-3118 (2001); Kashiwada Y. et al., J. Nat.Prod. 61:1090-1095 (1998); Kashiwada Y. et al., J. Nat. Prod.63:1619-1622 (2000); and Kashiwada Y. et al., Chem. Pharm. Bull.48:1387-1390 (2000) disclose dimethylsuccinyl betulinic acid anddimethylsuccinyl oleanolic acid. Esterification of the 3′ carbon ofbetulin with succinic acid produced a compound capable of inhibitingHIV-1 activity (Pokrovskii, A. G. et al., Gos. Nauchnyi Tsentr Virusol.Biotekhnol. “Vector,” 9:485-491 (2001)).

Published International Application No. WO 02/26761 discloses the use ofbetulin and analogs thereof for treating fungal infections.

There exists a need for new HIV inhibition methods that are effectiveagainst drug resistant strains of the virus. The strategy of thisinvention is to provide therapeutic methods and compounds that inhibitthe virus in different ways from approved therapies.

The compound and methods of the present invention have a novel mechanismof action and therefore are active against HIV strains that areresistant to current reverse transcriptase and protease inhibitors. Assuch, this invention offers a completely new approach for treatingHIV/AIDS.

BRIEF SUMMARY OF THE INVENTION

Generally, the invention provides methods of inhibiting, inhibitorycompounds and methods of identifying inhibitory compounds that targetproteolytic processing of the HIV-1 Gag protein. In one embodiment, suchcompounds may directly or indirectly inhibit the interaction of aprotease enzyme with HIV-1 Gag protein. In another embodiment, suchinhibition of interaction occurs via the binding of a compound to Gag.The inhibition of protease cleavage of the CA-SP1 protein of HIV-1 Gagby 3-O-(3′,3′-dimethylsuccinyl)betulinic acid (DSB) is one example, butother proteolytic cleavage sites can be targeted by a similar approachusing inhibitory compounds that interact with the substrate in a mannersimilar to that in which DSB interacts with Gag.

Another aspect of the invention is directed to a method of inhibitingthe processing of the viral Gag p25 protein (CA-SP1) to p24 (CA), buthaving no effect on other Gag processing steps.

A further aspect of the invention is directed to a method foridentifying compounds that inhibit processing of the viral Gag p25protein (CA-SP1) to p24 (CA), but have no effect on other Gag processingsteps.

In one aspect, the invention is drawn to a compound or pharmaceuticalcomposition identified by the method for identifying compounds thatinhibit HIV-1 replication disclosed herein.

In another aspect, the present invention is directed to a polynucleotidecomprising a sequence which encodes an amino acid sequence containing amutation in the Gag p25 protein, said mutation resulting in a decreasein the inhibition of processing of p25 to p24 by3-O-(3′,3′-dimethylsuccinyl)betulinic acid. This aspect of the inventionis also directed to a vector, virus and host cell comprising saidpolynucleotide, and a method of making said protein.

A further aspect of the present invention is directed to an amino acidsequence containing a mutation in the Gag p25 protein, said mutationresulting in a decrease in the inhibition of processing of p25 to p24 by3-O-(3′,3′-dimethylsuccinyl)betulinic acid.

An additional aspect of the invention is directed to an antibody whichselectively binds an amino acid sequence containing a mutation in theGag p25 protein, said mutation resulting in a decrease in the inhibitionof processing of p25 to p24 by 3-O-(3′,3′-dimethylsuccinyl)betulinicacid. Also included in this aspect of the invention are a method ofmaking said antibody, a hybridoma producing said antibody and a methodof making said hybridoma.

In a further embodiment, the invention is directed to a kit comprising apolynucleotide, polypeptide or antibody disclosed herein.

The invention further relates to a method of inhibiting HIV-1 infectionin cells of an animal by contacting said cells with a compound thatblocks the maturation of virus particles released from treated infectedcells. In one embodiment, the released virus particles exhibitnon-condensed cores and a distinctive thin electron-dense layer near theviral membrane and have reduced infectivity. A method is included ofcontacting animal cells with a compound that both inhibits processing ofthe viral Gag p25 protein and that disrupts the maturation of virusparticles. Also, included is a method of treating HIV-infected cells,wherein the HIV infecting said cells does not respond to other HIVtherapies.

This invention further includes a method for identifying compounds thatinhibit processing of the viral Gag p25 protein (CA-SP1) to p24 (CA),but have no significant effect on other Gag processing steps. The methodinvolves contacting HIV-1 infected cells with a test compound, andthereafter analyzing virus particles that are released to detect thepresence of p25. Methods to detect p25 include western blotting of viralproteins and detecting using an antibody to p25, gel electrophoresis,and imaging of metabolically labeled proteins. Methods to detect p25also include immunoassays using an antibody to p25 or SP1 (p2) or to anepitope tag inserted into the SP1 sequence.

The invention is further directed to a method for identifying compoundsinvolving contacting HIV-1 infected cells with a compound, andthereafter analyzing virus particles released by the contacted cells, bythin-sectioning and transmission electron microscopy, and determiningwhether viral particles with non-condensed cores and a distinctive thinelectron-dense layer near the viral membrane are present.

The invention is also directed to compounds identified by theaforementioned screening methods. In additional embodiments, theinvention is drawn to a method of treating HIV-1 infection in a patientby administering a compound that inhibits processing of the viral Gagp25 protein (CA-SP1) to p24 (CA), but does not significantly affectother Gag processing steps. In related embodiments, such inhibition maybe accompanied by a different observable phenotypes. For example,inhibition may not necessarily significantly reduce the quantity ofvirions released from treated infected cells; and/or said inhibition mayhave little or no significant effect on the amount of RNA incorporationinto the released virions; and/or said inhibition disrupts thematuration of virions released from infected cells treated with saidcompound. In related embodiments, the virion structure may be affected,and a majority of virions released from treated infected cells exhibitspherical, electron-dense cores that are acentric with respect to theviral particle; and/or possess crescent-shaped electron-dense layerslying just inside the viral membrane; and/or and have reduced or noinfectivity.

In additional embodiments, the invention is drawn to a method oftreating HIV-1 infection in a patient by administering a compound thatinhibits the interaction of HIV protease with CA-SP1, which results inthe inhibition of the processing of the viral Gag p25 protein (CA-SP1)to p24 (CA), but has no significant effect on other Gag processingsteps. Such inhibition may be direct or, alternatively, indirect; and/ormay involve said compound binding to the viral Gag protein such thatinteraction of HIV protease with CA-SP1 is inhibited. The invention isalso drawn to a method of treating HIV in a patient with a compound thatbinds at or near the site of cleavage of the viral Gag p25 protein(CA-SP1) to p24 (CA), thereby inhibiting the interaction of HIV proteasewith the CA-SP1 cleavage site and resulting in the inhibition ofprocessing of p25 to p24.

In other embodiments, the invention is drawn to a method of treatingHIV-1-infection in a patient by administering a compound that inhibitsprocessing of the viral Gag p25 protein (CA-SP1) to p24 (CA), whereinsaid compound binds to a polypeptide with an amino acid sequence havingat least about 40%, 50%, 60%, 70%, 80%, 90% identity, or which isidentical to a sequence selected from the group consisting of:

(SEQ ID NO: 21) (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPHKARILAEAMSQVTNSATIM; (SEQ ID NO: 22) (b)KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHK ARVLAEAMSQVTNPATIM; (SEQID NO: 23) (c) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ID NO: 24) (d)MTACQGVGGPGHKARVLAEAMSQVTKPATIM; (SEQ ID NO: 25) (e) SHKARILAEAMSQV and(SEQ ID NO: 26) (f) GHKARVLAEAMSQV.

In other embodiments, the invention is drawn to a method of treatingHIV-1-infection in a patient by administering a compound that inhibitsprocessing of the viral Gag p25 protein (CA-SP1) to p24 (CA), whereinsaid compound binds to a polypeptide encoded by a polynucleotidesequence having at least about 40%, 50%, 60%, 70%, 80%, 90% identity, orwhich is identical to a polynucleotide selected from the groupconsisting of: (a) about nucleotides 1243-1435 of; (b) about nucleotides1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ IDNO: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (e) aboutnucleotides 1370-1413 of SEQ ID NO: 18; and (f) about nucleotides1857-1899 of SEQ ID NO: 19.

In another aspect, the invention is drawn to a method of inhibitingprocessing of the viral Gag p25 protein (CA-SP1) by administration of acompound. In related embodiments, such a compound binds to a polypeptidewith an amino acid sequence having at least about 40%, 50%, 60%, 70%,80%, 90% identity, or which is identical to a sequence selected from thegroup consisting of:

(SEQ ID NO: 21) (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPHKARILAEAMSQVTNSATIM; (SEQ ID NO: 22) (b)KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHK ARVLAEAMSQVTNPATIM; (SEQID NO: 23) (c) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ID NO: 24) (d)MTACQGVGGPGHKARVLAEAMSQVTKPATIM; (SEQ ID NO: 25) (e) SHKARILAEAMSQV; and(SEQ ID NO: 26) (f) GHKARVLAEAMSQV.

In related embodiments; the invention is drawns to a method ofinhibiting processing of the viral Gag p25 protein (CA-SP1) byadministration of a compound wherein said compound binds to apolypeptide encoded by a polynucleotide sequence having at least about40%, 50%, 60%, 70%, 80%, 90% identity, or which is identical to apolynucleotide selected from the group consisting of:

(a) about nucleotides 1243-1435 of SEQ ID NO: 18; (b) about nucleotides1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ IDNO: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (e) aboutnucleotides 1370-1413 of SEQ ID NO: 18; and (f) about nucleotides1857-1899 of SEQ ID NO: 19.

The invention may be useful in the treatment of HIV in patients who arenot adequately treated by other HIV-1 therapies. Accordingly, theinvention is also drawn to a method of treating a patient in need oftherapy, wherein the HIV-1 infecting said cells does not respond toother HIV-1 therapies. In another embodiment, methods of the inventionare practiced on a subject infected with an HIV that is resistant to adrug used to treat HIV infection. In one application, the HIV isresistant to a protease inhibitor, a polymerase inhibitor, a nucleosideanalog, a vaccine, a binding inhibitor, an immunomodulator, or any otherinhibitor. In another embodiment, methods of the invention are practicedon a subject infected with an HIV that is resistant to a drug used totreat HIV infection is selected from the group consisting of zidovudine,lamivudine, didanosine, zalcitabine, stavudine, abacavir, nevirapine,delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir,nelfinavir, tenofovir, amprenavir, adefovir, atazanavir, fosamprenavir,hydroxyurea, AL-721, ampligen, butylated hydroxytoluene;polymannoacetate, castanospermine; contracan; creme pharmatex, CS-87,penciclovir, famciclovir, acyclovir, cytofovir, ganciclovir, dextransulfate, D-penicillamine trisodium phosphonoformate, fusidic acid,HPA-23, eflornithine, nonoxynol, pentamidine isethionate, peptide T,phenyloin, isoniazid, ribavirin, rifabutin, ansamycin, trimetrexate,SK-818, suramin, UA001, and combinations thereof.

Compounds of the invention are also useful as part of combination oftherapies. Accordingly, in one aspect the invention is drawn to a methodof treating HIV in a patient, wherein said patient is administered saidcompound in combination with at least one anti-viral agent. Anti-viralagents suitable include, but are not limited to: zidovudine, lamivudine,didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine,emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir,amprenavir, tenofovir, adefovir, atazanavir, fosamprenavir, hydroxyurea,AL-721, ampligen, butylated hydroxytoluene; polymannoacetate,castanospermine; contracan; creme pharmatex, CS-87, penciclovir,famciclovir, acyclovir, cytofovir, ganciclovir, dextran sulfate,D-penicillamine trisodium phosphonoformate, fusidic acid, HPA-23,eflornithine, nonoxynol, pentamidine isethionate, peptide T, phenyloin,isoniazid, ribavirin, rifabutin, ansamycin, trimetrexate, SK-818,suramin, UA001, enfuvirtide, gp41-derived peptides, antibodies to CD4,soluble CD4, CD4-containing molecules, CD4-IgG2, and combinationsthereof. In another embodiment, the patient is administered saidcompound in combination with an immunomodulating agent, anticanceragent, antibacterial agent, antifungal agent, or a combination thereof.

The invention is also directed to compounds. Such compounds are usefulin a method of treating patients infected with HIV; in a method forinhibiting processing of the viral Gag p25 protein (CA-SP1) to p24 (CA),or in a method for treating human blood and human blood products. Suchcompounds useful in the present invention include, but are not limitedto derivatives of dimethylsuccinyl betulinic acid or dimethylsuccinylbetulin, or is selected from the group consisting of3-O-(3′,3′-dimethylsuccinyl)betulinic acid,3-O-(3′,3′-dimethylsuccinyl)betulin,3-O-(3′,3′-dimethylglutaryl)betulin,3-O-(3′,3′-dimethylsuccinyl)dihydrobetulinic acid;3-O-(3′,3′-dimethylglutaryl)betulinic acid,(3′,3′-dimethylglutaryl)dihydrobetulinic acid, 3-O-diglycolyl-betulinicacid, 3-O-diglycolyl-dihydrobetulinic acid and combinations thereof.

Compounds of the invention may be used alone, or administered withadditional compounds, including zidovudine, lamivudine, didanosine,zalcitabine, stavudine, abacavir, nevirapine, delavirdine,emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir,amprenavir, tenofovir, adefovir, atazanavir, fosamprenavir, hydroxyurea,AL-721, ampligen, butylated hydroxytoluene; polymannoacetate,castanospermine; contracan; creme pharmatex, CS-87, penciclovir,famciclovir, acyclovir, cytofovir, ganciclovir, dextran sulfate,D-penicillamine trisodium phosphonoformate, fusidic acid, HPA-23,eflornithine, nonoxynol, pentamidine isethionate, peptide T, phenyloin,isoniazid, ribavirin, rifabutin, ansamycin, trimetrexate, SK-818,suramin, UA001, enfuvirtide, gp41-derived peptides, antibodies to CD4,soluble CD4, CD4-containing molecules, CD4-IgG2, and combinationsthereof; an antiviral, an immunomodulating agent, anti-cancer agent,antibacterial agent, an anti-fungal agent, or combinations thereof.

In further embodiments, the invention is directed to a method oftreating human blood products comprising contacting said blood productswith a compound that inhibits processing of the viral Gag p25 protein(CA-SP1) to p24 (CA). In one aspect, said compound does notsignificantly affect other Gag processing steps. In related embodimentsof this method, said inhibition does not significantly reduce thequantity of virions released from treated infected cell; and/or haslittle or no significant effect on the amount of RNA incorporation intothe released virions; and/or inhibits the maturation of virions releasedfrom infected cells treated with said compound; and/or affects viralmorphology. Such effects on viral morphology include, but are notlimited to: the virions released from treated infected cells to exhibitspherical, electron-dense cores that are acentric with respect to theviral particle; and/or possess crescent-shaped electron-dense layerslying just inside the viral membrane; and/or and have reduced or noinfectivity. In related embodiments, the method involves theadministration of the compound which inhibits the interaction of HIVprotease with CA-SP1, which results in the inhibition of the processingof the viral Gag p25 protein (CA-SP1) to p24 (CA) but has no significanteffect on other Gag processing steps. This may be via direct, orindirect inhibition of the interaction of HIV protease with CA-SP 1;and/or may involve said compound binds to the viral Gag protein suchthat interaction of HIV protease with CA-SP1 is inhibited; and/or saidcompound binds at or near the site of cleavage of the viral Gag p25protein (CA-SP1) to p24 (CA), thereby inhibiting the interaction of HIVprotease with the CA-SP1 cleavage site and resulting in the inhibitionof processing of p25 to p24.

In a further embodiment, the invention is drawn to a method of treatinghuman blood products comprising contacting said blood products with acompound that inhibits processing of the viral Gag p25 protein (CA-SP1)to p24 (CA), wherein said compound binds to a polypeptide with an aminoacid sequence having at least about 40%, 50%, 60%, 70%, 80%, 90%identity, or which is identical to a sequence selected from the groupconsisting of:

(SEQ ID NO: 21) (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPHKARILAEAMSQVTNSATIM; (SEQ ID NO: 22) (b)KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHK ARVLAEAMSQVTNPATIM; (SEQID NO: 23) (c) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ID NO: 24) (d)MTACQGVGGPGHKARVLAEAMSQVTKPATIM; (SEQ ID NO: 25) (e) SHKARILAEAMSQV; and(SEQ ID NO: 26) (f) GHKARVLAEAMSQV.

In a related embodiment, the invention is drawn to a method of treatinghuman blood products comprising contacting said blood products with acompound that inhibits processing of the viral Gag p25 protein (CA-SP1)to p24 (CA), wherein said compound binds to a polypeptide encoded by apolynucleotide sequence having at least about 40%, 50%, 60%, 70%, 80%,90% identity, or which is identical a polynucleotide selected from thegroup consisting of:

(a) about nucleotides 1243-1435 of SEQ ID NO: 18; (b) about nucleotides1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ IDNO: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (e) aboutnucleotides 1370-1413 of SEQ ID NO: 18; and (f) about nucleotides1857-1899 of SEQ ID NO: 19.

The invention also embodies methods for identifying compounds thatinhibit HIV-1 replication. Accordingly, the invention also includes amethod of identifying compounds that inhibit HIV-1 replication in cellsof an animal, comprising: contacting a Gag protein comprising a CA-SP1cleavage site with a test compound; adding a labeled substance thatselectively binds near the CA-SP1 cleavage site; and measuringcompetition between the binding of the test compound and the labeledsubstance to the CA-SP1 cleavage site. In further embodiments of thismethod, the compounds inhibits the interaction of HIV-1 protease with atarget site by binding to said target site.

These methods also include embodiments wherein the CA-SP1 cleavage siteregion is contained within a polypeptide fragment or recombinantpeptide; and/or wherein the labeled substance is a labeled antibodyspecific for CA-SP1, and measuring the change in the amount of labeledantibody bound to the protein in the presence of test compound comparedwith a control. Labels include, but are not limited to, an enzyme,fluorescent substance, chemiluminescent substance, horseradishperoxidase, alkaline phosphatase, biotin, avidin, electron densesubstance, radioisotope and a combination thereof.

The method of identifying compounds that inhibit HIV-1 replication incells of an animal also comprises, in one embodiment, measuring thechange in the amount of labeled 3-O-(3′,3′-dimethylsuccinyl)betulinicacid bound to the protein in the presence of test compound, comparedwith a control, and wherein the labeled substance is3-O-(3′,3′-dimethylsuccinyl)betulinic acid.

In an alternative embodiment, the invention comprises a method foridentifying compounds that inhibit HIV-1 replication in the cells of ananimal which comprises: contacting a polypeptide comprising a CA-SP1cleavage site, with a protease in the presence of a test compound.Preferably the protease is related to HIV-1 protease, or is HIVprotease. In one embodiment, the method comprises; contacting apolypeptide comprising a wild type CA-SP1 cleavage site, with a proteasein the presence of a test compound and also contacting a polypeptidecomprising a mutant CA-SP1 cleavage site or a protein comprising analternative protease cleavage site with HIV-1 protease in the presenceof the test compound, detecting the cleavage, and comparing the amountof cleavage of the native wild-type polypeptide to the amount ofcleavage of the mutant polypeptide or to amount of cleavage of theprotein comprising an alternative protease cleavage site. In a relatedaspect of this method, the wild-type CA-SP1 or mutant CA-SP1 oralternative protease cleavage site region is contained within apolypeptide fragment or recombinant peptide. In a further relatedaspect, the polypeptide is labeled with a fluorescent moiety and afluorescence quenching moiety, each bound to opposite sides of theCA-SP1 cleavage site, and wherein said detecting comprises measuring thesignal from the fluorescent moiety. In another related embodiment, thepolypeptide is labeled with two fluorescent moieties, each bound toopposite sides of the CA-SP1 cleavage site, and wherein said detectingcomprises measuring the transfer of fluorescent energy from one moietyto the other in the presence of the test compound. In a furtherembodiment, the effect of the test compound on cleavage of thepolypeptide is detected by measuring the amount of a labeled antibodythat is bound to SP1 or p24 (CA). In a related aspect, the labeledantibody that binds CA, or the antibody that binds SP1 is labeled with amolecule selected from the group consisting of enzyme, fluorescentsubstance, chemiluminescent substance, horseradish peroxidase, alkalinephosphatase, biotin, avidin, electron dense substance, radioisotope, andcombinations thereof.

The invention is also directed to a method for identifying compoundsthat inhibit HIV-1 replication in cells of an animal. In one embodiment,the method comprises: contacting a test compound with cells infectedwith wild-type virus isolates and with cells infected with virusisolates having significantly reduced sensitivity to3-O-(3′,3′-dimethylsuccinyl)betulinic acid; and selecting test compoundsthat are more active against the wild-type virus isolate compared withvirus isolates that have reduced sensitivity to3-O-(3′,3′-dimethylsuccinyl)betulinic acid. In another embodiment, themethod comprises contacting HIV-1 infected cells with a test compound;lysing the infected cells or the released viral particles to form alysate, and analyzing the lysate to determine whether cleavage of theCA-SP1 protein has occurred. In this latter embodiment, said analyzingmay comprise measuring the presence or absence of p25; and or performinga western blot of viral proteins and detecting p25 using an antibody top25; and/or performing a gel electrophoresis of viral proteins andimaging of metabolically labeled proteins; and/or performing animmunoassay. Such an immunoassay may be performed by any methods knownin the art, including, but not limited to:

(a) capturing p25 and p24 on a substrate using an antibody thatselectively binds p24; and

(b) detecting the presence or absence of p25 on the substrate by usingan antibody that selectively binds p25. The invention also includes suchmodifications of the above assay as would be obvious to one of ordinaryskill in the art.

In a further embodiment, the method of identifying a compound accordingto the invention comprises the use of an epitope tag sequence insertedinto SP1 and the selective detection of p25 is performed using anantibody to the epitope tag.

The invention is also directed to a method for identifying compoundsthat inhibit HIV-1 replication in the cells of an animal comprising:contacting HIV-1 infected cells with a test compound and thereafteranalyzing the virus particles using transmission electron microscopy.Such analysis includes for example, looking for the presence ofspherical cores that are acentric with respect to the viral particle;and/or having crescent-shaped, electron-dense layers lying just insidethe viral membrane.

In additional aspects, the invention is drawn to an isolatedpolynucleotide comprising a sequence which encodes an amino acidsequence containing a mutation in an HIV Gag p25 protein (CA SP1), saidmutation resulting in a decrease in inhibition of processing of p25(CA-SP1) to p24 (CA) by 3-O-(3′,3′-dimethylsuccinyl)betulinic acid(DSB). This inhibition of processing of p25 may be due to a decrease ininhibition of the interaction of HIV-1 protease with Gag; and/or adecrease in the binding of 3-O-(3′,3′-dimethylsuccinyl)betulinic acid toGag; and/or a decrease in the binding of DSB at or near the CA-SP1cleavage site of Gag. Suitable polynucleotides also include thoseencoding a mutation at or near the CA-SP1 cleavage site or in the SP1domain of CA-SP1; and/or those encoding a mutation at or near the aminoacid sequence G/SHKARV/ILAEAMSQV (SEQ ID NO: 1); and/or those encodingthe amino acid sequences GHKARVLVEAMSQV (SEQ ID NO: 2) or SHKARILAEAMSQV(SEQ ID NO: 3); and/or isolated polynucleotide which is selected fromthe group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQID NO: 9; and/or having at least about 95% identity to a polynucleotideselected from the group consisting of SEQ ID NO: 4, and SEQ ID NO: 6;and/or having at least about 80% identity to a polynucleotide selectedfrom the group consisting of SEQ ID NO: 8 and SEQ ID NO: 9; and/orhaving at least about 95% identity to a polynucleotide selected from thegroup consisting of SEQ NO: 5 and SEQ ID NO: 7; and/or having at leastabout 80% identity to a polynucleotide of SEQ ID NO: 10. In additionalembodiments, the polynucleotide having more than about 40%, 50%, 60%,70%, 80%, 90%, 95%, 99% identity or which is identical to thepolynucleotide sequences listed above.

The invention is also drawn to vectors comprising such polynucleotidesas described above; to a host cell comprising such a vector; and to amethod of producing a polypeptide comprising incubating the host cellcontaining such a vector in a medium and recovering the polypeptide fromsaid medium.

In one embodiment, the invention is directed to an antibody. Such anantibody may bind to a polypeptide with an amino acid sequence having atleast about 40%, 50%, 60%, 70%, 80%, 90% identity, or which is identicalto a sequence selected from the group consisting of:

(SEQ ID NO: 21) (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPHKARILAEAMSQVTNSATIM; (SEQ ID NO: 22) (b)KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHK ARVLAEAMSQVTNPATIM; (SEQID NO: 23) (c) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ID NO: 24) (d)MTACQGVGGPGHKARVLAEAMSQVTKPATIM; (SEQ ID NO: 25) (e) SHKARILAEAMSQV; and(SEQ ID NO: 26) (f) GHKARVLAEAMSQV.

In a further related embodiment, the invention is drawn to an antibodywhich binds to a polypeptide encoded by a polynucleotide sequence havingat least about 40%, 50%, 60%, 70%, 80%, 90% identity, or which isidentical to a polynucleotide with a sequence selected from the groupconsisting of: (a) about nucleotides 1243-1435 of SEQ ID NO: 18; (b)about nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828-1920 of SEQ IDNO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; and (f) aboutnucleotides 1857-1899 of SEQ ID NO: 19.

In one embodiment, the antibody binds to amino acids of the CA-SP1region of the HIV-1 Gag polypeptide, wherein said amino acids comprise:SHKARILAEAMSQV (SEQ ID NO: 25) or GHKARVLAEAMSQV (SEQ ID NO: 26).

In one embodiment, the invention is drawn to an antibody that inhibitsthe binding of 3-O-(3′,3′-dimethylsuccinyl)betulinic acid to the CA-SP1region of the Gag polypeptide.

The invention is also drawn to mutant HIV-1 viruses. In one suchembodiment, the invention is an isolated mutant recombinant HIV-1 virus,wherein the processing of the viral Gag p25 protein (CA-SP1) to p24 (CA)in said virus is not significantly inhibited by3-O-(3′,3′-dimethylsuccinyl)betulinic acid. In related embodiments, thisvirus is not inhibited by 3-O-(3′,3′-dimethylsuccinyl)betulinic acid. Inanother embodiment, 3-O-(3′,3′-dimethylsuccinyl)betulinic acid does notinhibit the interaction of protease with the Gag polypeptide in thisvirus. In another, the virus does not bind to3-O-(3′,3′-dimethylsuccinyl)betulinic acid. In further embodiments theinvention is drawn to viruses wherein the amino acids of the CA-SP1region are replaced with alternative amino acids, or amino acids areadded to the CA-SP1 region, or where amino acids are deleted. In oneembodiment, one or more amino acids are deleted from the AEAMSQV (aminoacid no. 8-14 of SEQ ID NO:26) amino acid sequence in the CA-SP1 region.

A mutant viruses may be used in the methods of the invention describedelsewhere herein. For example, such viruses are useful in a method ofidentifying a compound which inhibits processing of the viral Gag p25protein (CA-SP1) to p24 (CA), the method comprising comparing theability of said compound to inhibit HIV-1 replication compared with thereplication of a the mutant virus outlined above. Such inhibition may beexamined in a cell, or in an animal, or in vitro.

The invention is also drawn to non-HIV-1 retroviruses that are sensitiveto 3-O-(3′,3′-dimethylsuccinyl)betulinic acid. In some embodiment, saidretrovirus encodes a CA-SP1 polypeptide with an amino acid sequencecomprising the sequence AEAMSQV (amino acid no. 8-14 of SEQ ID NO: 26)at or near the CA-SP1 cleavage site. In another embodiment, theretrovirus encodes a CA-SP1 polypeptide with an amino acid sequencecomprising the sequence VLAEAMSQV (amino acid no. 6-14 of SEQ ID NO: 26)at or near the CA-SP1 cleavage site. In another embodiment, theretrovirus encodes a CA-SP1 polypeptide with an amino acid sequencecomprising the sequence GHKARVLAEAMSQV (SEQ ID NO: 26) at or near theCA-SP1 cleavage site; in another the retrovirus comprises the amino acidsequence having at least 60%, 70%, 80%, 90% identity or which isidentical to the sequence encoded by the polynucleotide of SEQ ID NO:26,SEQ ID NO: 90; SEQ ID NO: 92; SEQ ID NO: 94; SEQ ID NO: 96; or SEQ IDNO: 98; in another embodiment the retrovirus comprises the amino acidsequence having at least 60%, 70%, 80%, 90% identity or which isidentical to the sequence of SEQ ID NO: 91; SEQ ID NO: 93; SEQ ID NO:95; SEQ ID NO: 97; or SEQ ID NO: 99. In another embodiment, theretrovirus comprises the nucleic acid sequence having at least 70%, 80%,90% or which is identical to the sequence of SEQ ID NO: 90; SEQ ID NO:92; SEQ ID NO: 94; SEQ ID NO: 96; or SEQ ID NO: 98.

Retroviruses of this embodiment of the invention include, but are notlimited to HIV-2, HTLV-I, HTLV-II, SIV, avian leukosis virus (ALV),endogenous avian retrovirus (EAV), mouse mammary tumor virus (MMTV),feline immunodeficiency virus (FIV), Bovine immunodeficiency virus(BIV), caprine arthritis encephalitis virus (CAEV), Visna-maedi virus,or feline leukemia virus (FeLV).

In a related embodiment, the invention is drawn to a method of making arecombinant non-HIV-1 lentivirus sensitive to DSB. This methodcomprises: deleting from the genome of said lentivirus the nucleotideswhich corresponds nucleotides deleting from the genome of saidlentivirus the nucleotides which correspond to nucleotides 1370-1413from SEQ ID NO: 18, in HIV-1; and inserting nucleotides 1370-1413 fromSEQ ID NO: 18 or nucleotides 1857-1899 of SEQ ID NO: 19 into said regionof said non-HIV-1 lentivirus.

Examples of chimeric lentiviruses that were, are or may be constructedby this method are described in FIG. 10.

Such viruses may be used in the methods of the invention describedelsewhere herein. For example, such recombinant non-HIV-1 lentivirusesmay be used in a method of identifying a compound which inhibitprocessing of the viral Gag p25 protein (CA-SP1) to p24 (CA), the methodconsisting of comparing of the ability of said compound to inhibitreplication of a wild-type non-HIV-1 lentivirus with the DSB-sensitiverecombinant variant thereof. Such inhibition may occur in a cell; in ananimal; or in vitro.

The invention is also drawn to an animal model of lentivirus infectioncomprising a suitable non-human animal host infected with a lentivirussensitive to 3-O-(3′,3′-dimethylsuccinyl)betulinic acid. In such anembodiment, the lentivirus may include, but is not limited to SIV; FIV;EIAV; BIV; CAEV; and Visna-Maedi virus.

The invention is also drawn to isolated polypeptides. In one embodiment,the invention is drawn to a polypeptide containing a mutation in an HIVCA-SP1 protein, said mutation which results in a decrease in inhibitionof processing of p25 by 3-O-(3′,3′-dimethylsuccinyl)betulinic acid. In arelated embodiment, this polypeptide is encoded by a polynucleotide thatcontains a mutation located at or near the CA-SP1 cleavage site or inthe SP1 domain encoded by SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 10and/or is encoded by a polynucleotide selected from the group consistingof SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9; and/orcomprises a sequence that is selected from the group consisting ofGHKARVLVEAMSQV (SEQ ID NO: 2) or SHKARILAEVMSQV (SEQ ID NO: 3); and/oris encoded by an isolated polynucleotide which hybridizes understringent conditions to a polynucleotide selected from the groupconsisting of SEQ ID NO: 5, SEQ ID NO: 7, and 10; and/or is part of achimeric or fusion protein.

The invention is also drawn to antibodies which selectively bind to anamino acid sequence containing a mutation in an HIV CA-SP1 protein whichresults in a decrease in the inhibition of processing of p25 (CA-SP1) top24 (CA) by 3-O-(3′3′-dimethylsuccinyl)betulinic acid. In one suchembodiment, the antibody selectively binds to a mutation located at ornear the CA-SP1 cleavage site or in the SP1 domain of CA-SP1; inanother, the antibody selectively binds to a mutation comprising asequence that is selected from the group consisting of GHKARVLVEAMSQV(SEQ ID NO: 2) or SHKARILAEVMSQV (SEQ ID NO: 3); in another embodiment,the antibody selectively binds an amino acid sequence selected from thegroup consisting of SEQ ID NO: 2 and SEQ ID NO: 3.

In another embodiment, the invention is drawn to an antibody thatselectively binds SP1 but not CA-SP1; another that selectively bindsCA-SP1 but not CA; another that selectively binds CA but not CA-SP1; anda further antibody that selectively binds at or near the CA-SP1 cleavagesite.

The invention is also directed to a compound identified by any of themethods elucidated herein. In one embodiment, the compounds is not acompound selected from the group consisting of3-O-(3′,3′-dimethylsuccinyl)betulinic acid,3-O-(3′,3′-dimethylsuccinyl)betulin,3-O-(3′,3′-dimethylglutaryl)betulin,3-O-(3′,3′-dimethylsuccinyl)dihydrobetulinic acid,3-O-(3′,3′-dimethylglutaryl)betulinic acid,(3′,3′-dimethylglutaryl)dihydrobetulinic acid, 3-O-diglycolyl-betulinicacid, 3-O-diglycolyl-dihydrobetulinic acid, and combinations thereof.

The invention is also drawn to a pharmaceutical composition. In oneembodiment, the pharmaceutical composition comprises derivatives ofdimethylsuccinyl betulinic acid or dimethylsuccinyl betulin; in another,the pharmaceutical composition comprises a compound selected from thegroup consisting of 3-O-(3′,3′-dimethylsuccinyl)betulinic acid,3-O-(3′,3′-dimethylsuccinyl)betulin,3-O-(3′,3′-dimethylglutaryl)betulin,3-O-(3′,3′-dimethylsuccinyl)dihydrobetulinic acid,3-O-(3′,3′-dimethylglutaryl)betulinic acid,(3′,3′-dimethylglutaryl)dihydrobetulinic acid, 3-O-diglycolyl-betulinicacid, 3-O-diglycolyl-dihydrobetulinic acid, and combinations thereof. Inanother embodiment, the pharmaceutical composition comprises one or morecompounds identified according to the methods of the invention which arenot otherwise listed; or any pharmaceutically acceptable salt, ester orprodrug thereof, and a pharmaceutically acceptable carrier. In anotherembodiment, the pharmaceutical composition further comprising ananti-viral agent which may include any one of zidovudine, lamivudine,didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine,emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir,tenofovir, amprenavir, adefovir, atazanavir, fosamprenavir, hydroxyurea,AL-721, ampligen, butylated hydroxytoluene; polymannoacetate,castanospermine; contracan; creme pharmatex, CS-87, penciclovir,famciclovir, acyclovir, cytofovir, ganciclovir, dextran sulfate,D-penicillamine trisodium phosphonoformate, fusidic acid, HPA-23,eflornithine, nonoxynol, pentamidine isethionate, peptide T, phenyloin,isoniazid, ribavirin, rifabutin, ansamycin, trimetrexate, SK-818,suramin, UA001, combinations thereof, any other antiviral,immunomodulating agent, anti-cancer agent, anti-fungal agent,anti-bacterial agent, or combinations thereof.

The invention is also drawn to a method of determining if an individualis infected with HIV-1 that is susceptible to treatment by a compoundthat inhibits p25 processing. In one embodiment, the method involvestaking blood from the patient, genotyping the viral RNA and determiningwhether the viral RNA contains mutations in the sequence encoding theregion of the CA-SP1 cleavage site.

The invention is also drawn to a method of treating a disease in apatient in need thereof comprising:

identifying a compound which inhibits the processing of viral Gag p25protein (CA-SP1) to p24 (CA), but has no significant effect on other Gagprocessing steps;

obtaining regulatory approval for the sale and use of said compound;

packaging the compound for sale and treatment of a disease in a patientin need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. DSB does not disrupt the activity of HIV-1 protease at aconcentration of 50 μg/mL. In DSB-containing samples recombinant Gag isprocessed correctly. In contrast, indinavir blocks protease activity at5 μg/mL as evidenced by the absence of bands corresponding to p24 andthe MA-CA precursor.

FIG. 2. Western blots of virion-associated Gag derived from chronicallyinfected H9/HIV-1_(IIIB1), H9/HIV-2_(ROD), and H9/SIVmac251 in thepresence of DSB (1 μg/mL), indinavir (1 μg/mL) or control (DMSO). Gagproteins were visualized using HIV-Ig (HIV-1) or monkey anti-SIVmac251serum (HIV-2 and SIV; NIH AIDS Research and Reference Reagent Program).

FIG. 3. EM analysis of DSB-treated HIV-1 infected cells. The EM datashow two primary differences between DSB-treated and untreated samples.Virions generated in the presence of DSB are characterized by an absenceof conical, mature cores. In these samples the cores are uniformlyspherical and often acentric. Secondly, many virions display an electrondense layer inside the lipid bilayer but outside the core (indicatedwith arrows in the DSB-treated sample panels). In the DSB-treatedsamples no mature viral particles were observed.

FIG. 4 depicts amino acid sequences in the region of the CA-SP1 cleavagesite from DSB-sensitive HIV-1 isolates NL4-3 and RF (#1; SEQ ID NO: 1)and DSB-resistant HIV-1 isolates (#2; SEQ ID NO: 2 (NL4-3), and #3; SEQID NO: 3 (RF)). The differences between the native and DSB-resistantsequences involve an alanine to valine change at the first downstreamresidue (#2) and an alanine to valine change in the third downstreamresidue (#3) from the CA-SP1 cleavage site (−|−). These residues areunderlined and bolded for ease of identification.

FIG. 5 depicts the + sense consensus sequence for the A364VDSB-resistant NL4-3 mutant (SEQ ID NO: 4) beginning with the start ofgag and continuing into pol, including the entire protease codingregion. Missense mutations not found in the wild-type NL4-3 GENBANKM19921 sequence are in bold and gray shadowing. The coding sequence forthe consensus CA-SP1 cleavage site region is underlined. The shaded areaincluding the cleavage site denotes the SP1 sequence. The first mutationis the A364V mutation.

The second amino acid change (in protease) was also found in theparental clone and has been confirmed to correspond to a sequencingerror in the original GENBANK entry. Therefore, no mutations actuallyoccurred in protease.

FIG. 6 depicts the + sense consensus sequence for the DSB-sensitiveNL4-3 parental isolate (SEQ ID NO: 5) that was passaged in the absenceof drug in parallel with the A364V mutant isolate.

FIG. 7 depicts the + sense consensus sequence for the A366VDSB-resistant HIV-1 RF mutant (SEQ ID NO: 6) beginning with the start ofthe gag and continuing into pol, including the entire protease codingregion. Missense mutations not found in the wild-type HIV-1 RF GENBANKM17451 sequence are shadowed in gray. The region of the CA-SP1 cleavagesite is underlined. The only missense mutation not also found in theidentically passaged DSB-sensitive isolate is the A366V mutation in theCA-SP1 cleavage site.

FIG. 8 depicts the + sense consensus sequence for the DSB-sensitiveHIV-1 RF parental isolate (SEQ ID NO: 7), that was passaged in theabsence of drug in parallel with the A366V mutant isolate.

FIG. 9 depicts the polynucleotide sequences, SEQ ID NO: 8 and SEQ ID NO:9, which encode the polypeptides designated herein as SEQ ID NO: 2 andSEQ ID NO: 3, respectively. SEQ ID NO: 10 and 12 depict the nucleotidesequences that encode the parental polypeptide sequences designated asSEQ ID NO: 1. SEQ ID NO: 1 is a consensus sequence based on thesequences of the region from NL4-3 and RF

FIG. 10:

10A. Amino acid sequences in the CA-SP1 region of lentiviruses

10B: Amino acid sequences of the CA-SP1 region in HIV-1 strains RF andNL4-3

10C-10D: Nucleotide sequences of gag gene chimeric SIVs. The 42nucleotide sequence encoding the seven amino acids upstream and sevenamino acids downstream of the CA-SP1 cleavage site is underlined and inbold.

10E-H Nucleotide sequences of gag gene of chimeric FIV, EIAV and BIV tobe made according to the invention. The 42 nucleotide sequences encodingthe seven amino acids upstream and seven amino acids downstream of theCA-SP1 cleavage sites are underlined and in bold.

FIG. 11: Replication kinetics of PA-457 (DSB)-resistant mutants

FIG. 12: Sequential SP1 point deletions in the context of NL4-3 used toidentify residues necessary for DSB activity. The amino acid sequence ofSP1 domain in NL4-3 is shown. “Δ” indicates the deletion and “-” meansidentical residues between point deletion mutants and NL4-3

FIG. 13. Summary of particle production and infectivity of pointdeletions mutants.

FIG. 14. Western blots for viruses containing point deletions in SP1, inthe presence (+) and absence (−) of DSB.

FIG. 15. Substitution of HIV-1 CA-SP1 residues VL-AEAMSQV into SIVmac239backbone renders SIVmac239 sensitive to DSB.

(Top panel) Amino acid sequences near the CA-SP1 cleavage site(including entire SP1 region) are shown for SIVmac239, HIV-1 NL4-3 and aseries of SIV mutants into which various NL4-3 residues (underlined)were inserted. Dashes (“−”) indicates the residues are the same as thosein SIVmac239.

(Bottom panel) Western blots showing the CA and CA-SP1 proteins for thisseries of viruses in the presence (+) or absence (−) of DSB.

FIG. 16: Sequence conservation in the CA-SP1 region of Lentiviruses.

FIG. 16. Cloning Strategy: Substituting HIV-1 specific CA-SP1 residuesinto the corresponding Gag region of FIV, EIAV or BIV

FIG. 17. HIV-1 NL4-3 SP1 tagged with an epitope. Sequences of SP1peptides with peptide tags inserted are shown. “Δ” indicates deletedresidue and “-” indicates that the residue is identical to that in NL4-3SP1

FIG. 18A-C: HIV-1 strain RF polynucleotide sequence. The nucleotidesequence of the Gag polyprotein is underlined and in bold. The 42nucleotide sequence encoding the seven amino acids upstream and sevenamino acids downstream of the CA-SP1 cleavage site is highlighted ingreen. An additional 129 nucleotides (43 amino acid residues) upstreamof the cleavage site in CA and the remaining 21 nucleotides (seven aminoacids residues) in SP1 are highlighted.

FIG. 19A-E: HIV-1 strain NL4-3 polynucleotide sequence. The nucleotidesequence of the Gag polyprotein is underlined and in bold. The 42nucleotide sequence encoding the seven amino acids upstream and sevenamino acids downstream of the CA-SP1 cleavage site is highlighted ingreen. An additional 129 nucleotides (43 amino acid residues) upstreamof the cleavage site in CA and the remaining 21 nucleotides (seven aminoacids residues) in SP1 are highlighted.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods of inhibiting HIV-1replication in the cells of an animal. More specifically, the inventioninvolves methods of inhibiting HIV-1 replication in the cells of amammal by contacting infected cells with a compound that inhibits theprocessing of the viral Gag p25 protein (CA-SP1) to the p24 protein(CA). More specifically, such compounds inhibit the processing of theviral Gag p25 protein (CA-SP1) to the p24 protein (CA) withoutsignificantly affecting other Gag processing steps.

“A compound that does not significantly affect other Gag processingsteps” means that the compound in question predominantly inhibitsprocessing of p25 to p24, but does not necessarily preclude thepossibility of having additional minor effects on other Gag processingsteps.

“Significant” or “Significantly,” where not otherwise defined herein,means an observable or measurable change compared to the process in theabsence of a compound. However, not all observable or measureablechanges may necessarily be significant.

A number of viral phenotypes may also be observed in practicing themethod of the invention. One result of contacting an infected cell withthe compounds of the invention may be the formation of noninfectiousviral particles. Alternatively, or in addition, contacting infectedcells with a compound that inhibits p25 to p24 processing, results inthe formation of non-infectious viral particles, but where there is nosignificant effect on other Gag processing steps. This may notsignificantly reduce the quantity of virus released from treated cellsand/or has no little or no significant effect on the amount of RNAincorporation into the released virions.

Accordingly, the invention is also drawn to a method of inhibiting HIVinfection in cells of an animal comprising contacting said cells with acompound that inhibits p25 processing and also affects other viralphenotypes, described above.

Mutant viruses defective in CA-SP1 cleavage have been shown to benon-infectious (Wiegers K. et al., J. Virol. 72:2846-2854 (1998)).3-O-(3′,3′-dimethylsuccinyl)betulinic acid (DSB) is an example of acompound that disrupts p25 to p24 processing and potently inhibits HIV-1replication. This compound's activity is specific for the p25 to p24processing step, not other steps in Gag processing. Furthermore, DSBtreatment results in the aberrant HIV particle morphology as describedin FIG. 3.

Identification of HIV-1 Determinants Associated with Sensitivity to3-O-(3′,3′-dimethylsuccinyl)betulinic Acid

Generation and Selection of HIV-1 Viruses Resistant to DSB.

Mutant forms of HIV-1 have been generated in which the amino acidsequence in the region of the CA-SP1 cleavage site is modified,decreasing the sensitivity of these strains to compounds that disruptCA-SP1 processing. Data on these mutant viruses have been used toidentify the amino acid residues in wild-type Gag that are implicated inthe antiviral activity of these compounds. In one embodiment, compoundsthat disrupt CA-SP1 processing directly or indirectly inhibit theinteraction of HIV-1 protease with the region of the Gag proteincontaining these amino acid residues. In another embodiment, compoundsthat disrupt CA-SP1 processing bind to the region containing these aminoacid residues. As used herein, the terms “bind,” “bound” or “binding”refers to binding or attachment including, e.g., ionic interactions,electrostatic hydrophobic interactions, hydrogen bonds, etc; and alsoincludes associations that may be covalent, e.g., by chemicallycoupling. Covalent bonds can be, for example, ester, ether,phosphoester, thioester, thioether, urethane, amide, amine, peptide,imide, hydrazone, hydrazide, carbon-sulfur bonds, carbon-phosphorusbonds, and the like. The term “bound” is broader than and includes termssuch as “coupled,” “conjugated” and “attached.”

In another embodiment, compounds that disrupt CA-SP1 processing bind toanother region of Gag and thereby inhibit the interaction of HIV-1protease with the region of the CA-SP1 cleavage site. In anotherembodiment, viruses or recombinant proteins that contain mutations inthe region of the CA-SP1 cleavage site can be used in screening assaysto identify compounds that disrupt CA-SP1 processing.

In one set of experiments, amino acid residues in HIV-1 Gag that areinvolved in the disruption of CA-SP1 processing by3-O-(3′,3′-dimethylsuccinyl)betulinic acid (DSB) were identified bysequencing the gag-pol gene of virus isolates that had been selected forresistance to DSB. The amino acid sequences from these resistant viruseswere compared with the gag-pol gene sequences from DSB-sensitive HIV-1isolates. Two single amino acid changes were identified in theDSB-resistant viruses, an alanine (Ala) to valine (Val) substitution atresidue 364 (SEQ ID NO: 4) and in a second isolate, at residue 366 (SEQID NO: 6), in the Gag polyprotein (see FIG. 4). These residues arelocated immediately downstream of the CA-SP1 cleavage site (at theN-terminus of SP1). Alanine is highly conserved at these positionsthroughout all HIV-1 subtypes listed in the Los Alamos NationalLaboratory database. The five amino acid residues upstream anddownstream of the CA-SP1 cleavage site are also highly conserved amongthe various subtypes. However, isoleucine replaces valine at theposition two residues upstream of the cleavage site in a number ofclades (c.f., FIG. 4, SEQ ID NO. 1). (“HIV Sequence Compendium 2002,”Kuiken et al. eds. Los Alamos National Laboratory, Los Alamos, N. Mex.)

In order to more extensively map the viral genetic determinants for DSBresistance, additional experiments were performed to select for virusesin vitro that are drug resistant. Multiple parallel cultures of Jurkat Tcells (5×10⁵ each) were transfected with the proviral DNA clone pNL4-3in the presence or absence of 10-50 ng/ml DSB. The cells were passagedevery two days, and fresh drug was added at each passage. Virusreplication was monitored by measuring reverse transcriptase activity inculture supernatants. Virus was isolated from culture supernatantsharvested at selected timepoints, and genomic DNA was amplified byRT-PCR using primers that spanned the coding region between theN-terminus of CA and the N-terminus of RT. The amplified product wasthen sequenced using the same set of primers.

In one experiment, an A366V mutation was identified in the SP1 region ofNL4-3 virus cultured in the presence of DSB (note: numbering is relativeto the Gag polyprotein). Upon further passaging, a double mutant wasidentified that contained a G357S mutation in CA as well as the A366Vmutation in SP1. The A366V mutation was identified previously inexperiments selecting for resistant variants of the RF isolate.Interestingly, the wild-type RF sequence also contains a serine residueat position 357 in CA (FIG. 4). Since serine is present at this positionin isolates (such as RF) that are sensitive to DSB, the CA G357Smutation alone is not sufficient to confer resistance to DSB. Todetermine the contribution of each of these mutations to drugresistance, the A366V mutation and the A366V/G357S double mutation werere-engineered into the wild-type NL4-3 backbone by site-directedmutagenesis. The resulting constructs were transfected into Jurkat Tcells and characterized in a virus replication assay as described abovefor the selection of resistance. SDS-PAGE analysis of transfected celllysates and virus released into the media demonstrated that the A366Vmutant Gag was processed and released from cells inefficiently (data notshown) and thus replicated very poorly even in the absence of drug (FIG.11) However, the A366V/G357S double mutant replicated efficiently in theabsence or presence of DSB. There data indicate that the resistantmutant, A366V, requires a serine at the 357 position in the CA region ofGag to compensate for a deleterious effect on virus replication (FIG.11).

In a further experiment, ten different resistant isolates weregenerated. Sequencing of these isolates identified four additionalmutations not previously seen in resistance selection experiments. Thesewere H358Y, L363F and L363M in CA, and A402T in the NC region of Gag.None of these mutations are present in the consensus sequences for HIV-1clades A-O, reflecting the breadth of activity of DSB againstgenetically diverse clades of HIV-1. The L363M substitution in CA wasfound in the consensus sequence for HIV-2, which may, in part, explainthe specificity of DSB for HIV-1.

These results demonstrate the presence of specific genetic determinantsfor DSB activity in HIV-1, and that these determinants are centeredaround the CA-SP1 cleavage site.

HIV-1 NL4-3 Deletion and SIV Insertion Studies Used to Identify ViralGenetic Determinants of DSB Sensitivity

Results from in vitro resistance selection experiments indicated thatthe determinants of DSB HIV-1 inhibitory activity map to the region ofGag flanking the CA-SP1 cleavage site. In order to better define theviral genetic determinant for DSB, HIV-1 point-deletion mutagenesis andSIV insertion studies were undertaken to identify the specific aminoacid residues associated with compound activity. The study was carriedout as follows. Single residue deletions starting with residue E365 andcontinuing through residue M377 were engineered into the SP1 domain ofthe infectious HIV-1 molecular clone NL4-3 (FIG. 12). The effect ofthese point deletions on viral particle production, infectivity, Gagprocessing and sensitivity to DSB was determined. The results of theseexperiments were used to identify the Gag residues in the region of theCA-SP1 cleavage site that are associated with DSB activity. The residuesassociated with activity were inserted into the CA-SP1 cleavage siteregion of the DSB-resistant virus SIV (Mac 239 isolate) to generate aHIV-1, SIV chimeric virus (SHIV). Point substitution of HIV-1 residuesfrom the N-terminus of the CA protein were made into this chimeric virusuntil the minimal sequence necessary to rescue DSB activity wasidentified. This minimal sequence necessary to gain DSB activity isconsidered a primary viral genetic determinant of DSB activity. It maysuggest the molecular determinant of DSB activity.

Methods:

Construction of NL4-3 Single Point-Deletion Mutants.

Single point-deletion constructs were generated using thePCR-ligation-PCR (PLP) strategy as previously described. HIV-1 NL4-3plasmid DNA was used as the template to perform all PCR reactions forgenerating point deletions spanning the complete Gag SP1 domain with theexception of the first residue of SP1.

ΔE365 was generated using NL4-3 as the template with Vent DNA polymerase(NEB) by using deletion-specific downstream primer (Primer 1) withuniversal upstream primer (Primer 2) (Table 1). The fragment derivedfrom this was termed as a first flanking PCR fragment. A second flankingfragment was amplified using deletion-specific upstream primer (Primer.3) and universal downstream primer (Primer 4) (Table 1). To generateother deletion constructs (ΔA366, ΔM367, ΔS368, ΔQ369, ΔV370, ΔT371,ΔN372, ΔP373, ΔA374, ΔT375, ΔI376, and ΔM377). PCR procedures weresimilarly performed by varying deletion-specific downstream and upstreamprimers corresponding to each specific point deletion (Table 1).

Each of these parallel two adjacent PCR fragments was gel purified,phosphorylated using T4 polynucleotide kinase (NEB), and ligated byusing T4 DNA ligase (NEB). After inactivation at 65° C. for 15 minutes,the ligation reaction was used for a subsequent amplification withuniversal upstream primer (Primer. 2) and downstream primer (Primer. 4).This product was gel purified, digested with SpeI and ApaI, and thenligated into the SpeI and ApaI sites of NL4-3 proviral DNA clone.

Standard PCR conditions were used for the above-described reactions.These included, one cycle of denaturation at 95° C. for 1 minutes 30seconds, followed by 30 cycles of denaturation at 95° C. for 30 seconds,60° C. for 30 seconds and 72° C. for 30 seconds. The PCR reactions wereset up using the following components: δ5 μL 10×NEB Thermophilic buffer

2 μL 10 mM dNTPs

1 μL 10 nM MgSO₂

1 μL 50 μmol upstream primer

1 μL 50 μmol downstream primer

1 μL 50 ng/mL template DNA

0.5 μL Vent DNA polymerase

38.5 μL ddH₂O

A 10 μL aliquot was run on a 1.0% agarose gel to make sure the correctsize product was amplified. The PCR products were then gel isolated andpurified with a Qiaex II gel extraction Kit (Qiagen). The gel-purifiedtwo adjacent PCR fragments were individually phosphorylated in thefollowing reaction by using T4 polynucleotide kinase (NEB) prior toligation. The phosphorylation reaction was set up as follows:

2 μL 10×T4 polynucleotide kinase buffer

2 μL 10 mM ATP

1 μL T4 polynucleotide kinase

15 μL gel purified DNA of each of these two adjacent PCR fragments Thereaction was incubated at 37° C. for 1 hour. Following the inactivationat 65° C. for 10 minutes, the adjacent phosphorylated PCR fragments werethen ligated together by using T4 DNA ligase (NEB) under followingconditions:

3 μL 10×T4 DNA ligase buffer

13 μL of each of two adjacent PCR fragments

1 μL T4 DNA ligase

After overnight incubation at 16° C. the ligation reaction product wasused in a second round PCR reaction to amplify the full-length PCRfragment spanning these two adjacent PCR products. The second round PCRreaction was performed as described above with the exception that onlyuniversal upstream primer (Primer. 2) and downstream primer (Primer. 4)were used. Again, a 10 μL aliquot was run on a agarose gel to make surethe correct product was amplified. The full-length PCR fragments werethen gel isolated and purified using a Qiaex II kit. The purifiedfull-length PCR fragment, together with NL4-3, were then cut with SpeIand ApaI under the following conditions:

2 μL 10×NE buffer 4 (NEB)

1 μL ApaI (NEB)

1 μL SpeI (NEB)

16 μL full length PCR product (1 μg) or NL4-3 (500 ng)

The above restriction enzyme digestion mixture was incubated at 37° C.for 2 hours. Digested DNA fragments for the full-length PCR product andthe NL4-3 plasmid were individually gel isolated and purified using aQiaex II kit. The digested vector NL4-3 and full length PCR fragmentwere ligated using T4 DNA ligase under the following procedure:

1 μL 10×T4 DNA ligase buffer

1 μL (25-50 ng) digested NL4-3 vector

7 μL digested (200 ng-400 ng) digested PCR fragment (700 bp)

1 μL T4 DNA ligase

The ligation reaction was incubated at 16° C. overnight and the ligatedproducts were transformed into Escherichia coli Max Efficiency Stb12(Invitrogen) by heat shock according to instruction (Invitrogen). Theproviral DNA clones were then screened by automatically sequencing usinga Taq Dye Deoxy Terminator cycle Sequencer Kit (Applied Biosystems)individually using internal primers (Primer 29 and 30) Following theverification the mutations the proviral DNA clones were used for variousfuture studies.

Construction of SIV Chimeric Mutants

A panel of SIV chimeric constructs harboring various residues of NL4-3CA-SP1 boundary region was generated using the SIVmac239 molecular cloneby employing PCR and cloning procedures described above. Theseconstructs and their amino acid sequences in the CA-SP1 boundary regionare shown in FIG. 15. SIV mac239 was used to generate the SIV DD and DEconstructs. The SIV DD construct was used to generate SIV DM. DifferentSIV chimeric constructs were produced in the PCR by varying respectivemutagenic upstream and downstream primers corresponding to each chimera(Table 1). Each of these parallel two adjacent PCR fragments was gelpurified and directly used without phosphohorylation treatment for asubsequent amplification with universal upstream primer (Primer. 31) anddownstream primer (Primer 32). This product was gel purified, digestedwith BamHI and SbfI, and then ligated into the BamHI and SbfI sites ofSIVmac239 proviral DNA clone. The proviral DNA clones were then screenedby automatically sequencing using a Taq Dye Deoxy Terminator cycleSequencer Kit (Applied Biosystems) individually using an internal primer(Primer. 39). Following the verification the mutations the proviral DNAclones were used for various future studies.

Cell Culture and DNA Transfection

HeLa cells were maintained in DMEM (Invitrogen) (10% FBS, 100 U/mlpenicillin, and 100 μg/ml Streptomycin) and passaged upon confluence.Jurkat cells were cultured in RPMI 1640 (Invitrogen) (10% FBS, 100 U/mlpenicillin, and 100 μg/ml Streptomycin) and passaged every two or threedays.

To characterize the effect of deletion or substitution on viral particleproduction and Gag polyprotein processing, wild-type HIV-1 NL4-3 orSIVmac239 and respective mutant proviral DNAs were transfected into HeLacells by employing FuGENE 6 transfection reagent (Roche). Briefly, cellswere seeded into a 6-well plate (Corning) at a concentration of 0.5×105per well the day before transfection to reach 60 to 80% confluence onthe day of transfection. For each transfection, 3 μl of FuGENE 6 wasdiluted into 100 μl of serum-free DMEM followed by the addition of 1 μgof DNA. After gently mixing, the mixture of DNA-lipid complexes wasgently added drop wise into the cells containing 2 ml of complete DMEMmedium. Twenty-four hours post-transfection, medium containingDNA-FuGENE 6 complexes was removed, 2 ml of fresh DMEM was added intothe transfected cells. At 48 h post-transfection, medium containingviral particles was collected and clarified by centrifugation at 2,000rpm at 4° C. for 20 min in a Sorvall RT 6000B centrifuge. Virusparticle-containing supernatants were then concentrated through a 20%sucrose cushion in a microcentrifuge at 13,000 rpm at 4° C. for 120 minand pellets were resuspended in a lysis buffer (150 mM Tris-HCl, 5%Triton X-100, 1% deoxycholate, pH 8.0). The level of viral particleproduction for wild type NL4-3 and point deletion mutants was determinedby p24 antigen capture ELISA (ZeptoMetrix, Buffalo, N.Y.).

To examine the effect of deletion or substitution on Gag polyproteinprocessing (in the absence of DSB), SDS-PAGE and Western-Blot wasperformed. In brief, viral proteins were separated on a 12% NuPAGEBis-Tris Gel (Invitrogen) and transferred to a nitrocellulose membrane(Invitrogen) followed by blocking in a PBS buffer containing 0.5% Tweenand 5% dry milk. The membrane was incubated with immunoglobulin fromHIV-1-infected patients (HIV-Ig) (NIH AIDS research and referencereagent program) and hybridized with goat anti-human horseradishperoxidase (Sigma). For the membrane containing SIV proteins, themembrane was incubated with a reference polyclonal immune serum from aSIV-infected monkey (NIH AIDS Research and Reference Reagent Program)and hybridized with goat-anti-monkey horseradish peroxidase (Sigma). Theimmune complex was visualized with an ECL system (Amersham PharmacisBiotech) according to the instructions provided by the manufacturer.

To address the effect of deletion or substitution on the ability of DSBto inhibit CA-SP1 processing, HeLa cells were transfected with wild-typeHIV-1 NL4-3 or SIVmac239 and respective mutant proviral DNAs byemploying the procedure described above. DSB at a concentration of 1μg/ml and DMSO control were maintained throughout the entire culture andSDS-PAGE/Western-Blot for analyzing viral proteins derived from thesetransfections were performed as described in the previous paragraph.

The 50%-Tissue Culture Infectious Dose (TCID₅₀) per ml was used as ameasure of the infectivity of each deletion mutant. Mutant virusesderived from transfections in HeLa cells were used to infect U87CD4.CXCR4 cells. Each virus stock was tested in triplicate at a startingdilution of 1:10, followed by four-fold serial dilutions. Cells wereplated the day before infection at a density of 3×10³ cells/well. On theday of infection, culture media was removed from the cell plate and 90μl of diluted virus was added. On days 1, 3, and 6 post infection, viruswas removed from plate and 200 μl of culture media was added. On days 6and 8 post infection, supernatant was collected for p24 ELISA analysis.The virus dilution that caused 50% of the culture to be infected(TCID₅₀) was determined according to the method of Reed and Muench(Aldovini A. and B. Walker 1990; Dulbecco R. 1988).

Results

Viruses containing sequential point deletions within the Gag SP1 domain(FIG. 12) were characterized for particle production, infectivity, Gagprocessing and sensitivity to DSB. The results from these experimentswere used to identify SP1 residues associated with DSB activity.

As expected, the effect of point deletions on viral particle productionvaried as a function of the proximity of the change from the proteolyticcleavage site. The results from these experiments are summarized in FIG.13. Viruses with deletions at residues E366, A367 and M368 were mostaffected, generating <25% the number of particles normally observed inwild-type virus infection. In vitro infectivity assays were used tocharacterize the ability of the deletion mutants to support virusreplication. These experiments indicated that deletion of singleresidues at any of the five positions E365 through Q369 resulted in avirus that was either non-infectious or significantly impaired forreplication (FIG. 13). In contrast, starting with residue V370 andextending away from the CA-SP1 cleavage site, none of the characterizedpoint deletions resulted in a decrease in virus infectivity (FIG. 13).With the exception of viruses with deletions at positions I376 and M377all mutant viruses exhibited a normal or near normal Gag processingphenotype (FIG. 13). The results from these three sets of experimentspermitted the design and interpretation of experiments to identify thegenetic determinants of DSB activity.

Sensitivity to DSB was determined in experiments that characterized theeffect of DSB on a late step in Gag processing, CA-SP1 cleavage.Specifically, these assays measured the ability of DSB to disrupt CA-SP1processing. As seen, e.g. Example 8, the DSB-induced defect in Gagprocessing correlates with the ability of the compound to inhibit virusreplication. Results from these experiments indicate that deletion of asingle residue at any of the six positions E365 through V370significantly reduces the affect of DSB on CA-SP1 processing (FIG. 14).In contrast, starting with residue T372 and extending away from theCA-SP1 cleavage site, all of the characterized point deletions are fullysensitive to DSB-induced disruption of CA-SP1 processing (FIG. 14).

The SP1 residues associated with DSB activity consist of the contiguousresidues E365 through V370.

Residues A364 through V370 were inserted into the analogous position ofthe Gag SP1 domain in the DSB-resistant retrovirus SIV (Mac 239isolate). Additionally, the N-terminus of the CA protein of thischimeric virus was modified by cumulative substitution of residues foundin SIV with HIV-1-specific residues. This approach is summarized in FIG.15. Next, the effect of DSB on the Gag processing phenotype of each ofthe chimeric viruses was determined. As shown in FIG. 15, the SIV.DMvirus displays a Gag processing phenotype indicative of sensitivity toDSB. Thus, the minimum sequence of HIV-1 CA-SP1-specific residues thatneeds to be inserted to rescue DSB activity in the SHIVs extends fromV362 to V370

TABLE 1 PCR Mutagenesis Primers Primer PCR ID Sequence (5′ to 3′)Construct application  1 agccaaaactcttgctttatggcc ΔE365 First PCR (SEQID NO: 37) fragment (with No. 2 primer)  2 agtcagtgtggaaaatctctagcagtggAll All first (SEQ ID NO: 38) deletion PCR constructs fragments of NL4-3 3 gcaatgagccaagtaacaaatcca ΔE365 Second PCR (SEQ ID NO: 39) fragment(with No. 4 primer)  4 aggtatggtaaatgcagtatacttcctgaag All All second(SEQ ID NO: 40) deletion PCR constructs fragments  5ttcagccaaaactcttgctttatggcc ΔA366 First PCR (SEQ ID NO: 41) fragmentwith No. 2 primer)  6 atgagccaagtaacaaatccagc ΔA366 Second PCR (SEQ IDNO: 42) fragment (with No. 4 primer)  7 tgcttcagccaaaactcttgc ΔM367First PCR (SEQ ID NO: 43) fragment (with No. 2 primer)  8agccaagtaacaaatccagct ΔM367 Second PCR (SEQ ID NO: 44) fragment (withNo. 4 primer)  9 cattgcttcagccaaaactcttgc ΔS368 First PCR (SEQ ID NO:45) fragment (with No. 2 primer) 10 caagtaacaaatccagctacca ΔS368 SecondPCR (SEQ ID NO: 46) fragment (with No. 4 primer) 11gctcattgcttcagccaaaactctt ΔQ369 First PCR (SEQ ID NO: 47) fragment (withNo. 2 primer) 12 gtaacaaatccagctaccataa ΔQ369 Second PCR (SEQ ID NO: 48)fragment (with No. 4 primer) 13 acaaatccagctaccataatgatac ΔV370 FirstPCR (SEQ ID NO: 49) fragment (with No. 2 primer) 14ttggctcattgcttcagccaaaactc ΔV370 Second PCR (SEQ ID NO: 50) fragment(with No. 4 primer) 15 tacttggctcattgcttcagccaa ΔT371 First PCR (SEQ IDNO: 51) fragment (with No. 2 primer) 16 aatccagctaccataatgatacag ΔT371Second PCR (SEQ ID NO: 52) fragment (with No. 4 primer) 17tgttacttggctcattgcttc ΔN372 First PCR (SEQ ID NO: 53) fragment (with No.2 primer) 18 ccagctaccataatgatacagaaa ΔN372 Second PCR (SEQ ID NO: 54)fragment (with No. 4 primer) 19 atttgttacttggctcattgcttc ΔP373 First PCR(SEQ ID NO: 55) fragment (with No. 2 primer) 20gctaccataatgatacagaaaggcaa ΔP373 Second PCR (SEQ ID NO: 56) fragment(with No. 4 primer) 21 tggatttgttacttggctcattgc ΔA374 First PCR (SEQ IDNO: 57) fragment (with No. 2 primer) 22 accataatgatacagaaaggc ΔA374Second PCR (SEQ ID NO: 58) fragment (with No. 4 primer) 23agctggatttgttacttggctc ΔT375 First PCR (SEQ ID NO: 59) fragment (withNo. 2 primer) 24 ataatgatacagaaaggcaattttagg ΔT375 Second PCR (SEQ IDNO: 60) fragment (with No. 4 primer) 25 ggtagctggatttgttacttg ΔI376First PCR (SEQ ID NO: 61) fragment (with No. 2 primer) 26atgatacagaaaggcaattttaggaacc ΔI376 Second PCR (SEQ ID NO: 62) fragment(with No. 4 primer) 27 tatggtagctggatttgttac ΔM377 First PCR (SEQ ID NO:63) fragment (with No. 2 primer) 28 atacagaaaggcaattttagg ΔM377 SecondPCR (SEQ ID NO: 64) fragment (with No. 4 primer) 29 ccacctatcccagtaggagSequencing For NL4-3 (SEQ ID NO: 65) primer mutants 30ggcacagcaagcagcagctg Sequencing For NL4-3 (SEQ ID NO: 66) primer mutants31 gtagaccaacagcaccatctagcggcaga All For SIV (SEQ ID NO: 67)substitution mutants constructs of SIV 32 ggtaaagtaaaggcagtgtactgcctaaAll For SIV (SEQ ID NO: 68) substitution mutants constructs of SIV 33cactggtgcgaggacctgactcatggcttctgccatt SIV DD First PCR (SEQ ID NO: 69)fragment (with No. 31 primer) 34 aatggcagaagccatgagtcaggtcctcgcaccagtgSIV DD Second PCR (SEQ ID NO: 70) fragment (with No. 32 primer) 35ggcttctgccagtactctagccttctgt SIV DE First PCR (SEQ ID NO: 71) fragment(with No. 31 primer) 36 acagaaggctagagtactggcagaagcc SIV DE Second PCR(SEQ ID NO: 72) fragment (with No. 32 primer) 37ggcttctgccagtactctagccttctgt SIV DM First PCR (SEQ ID NO: 73) fragment(with No. 31 primer) 38 acagaaggctagagtactggcagaagcc SIV DM Second PCR(SEQ ID NO: 74) fragment (with No. 32 primer) 39atccaactggggttgcaaaaatgtg Sequencing For SIV (SEQ ID NO: 75) primermutant

The resistance and mutagenesis data presented above suggest that theGHKARVL-AEAMSQV amino acid sequence in the region of the HIV-1 GagCA-SP1 cleavage site serves as a genetic determinant of viralsensitivity to DSB.

Extending the Determinants of PA-457DSB Sensitivity to OtherLentiviruses CA-SP1 Chimeras as Animal Efficacy Models for Developmentof Maturation Inhibitors

The development of anti-HIV therapeutics has been hindered by the lackof an animal efficacy model. This lack of an animal model is primarilydue to the inability of most HIV-1 strains to replicate and causedisease in non-human primates. In some instances this problem has beenovercome through the use of chimeric viruses that incorporate theregion(s) of interest from the HIV-1 viral target into an SIV viralbackbone that will support replication in a non-human primate. The mostnotable example of this approach involves the HIV-1/SIV (SHIV) chimericviruses in which the proteins making up the infectious virus areexclusively SIV in origin with the exception of Env (gp120/gp41) whichis derived form HIV-1. These SHIV envelope chimeras have been usedextensively in HIV-1 vaccine development.

HIV-1 maturation inhibitors disrupt Gag CA-SP1 processing, which resultsin the formation and release of non-infectious viral particlesexhibiting aberrant core morphology. See e.g. Li et al. Proc Natl AcadSci USA. 100:13555-60 (2003). The betulinic acid derivative PA-457 DSBis an example of this class of inhibitors. The viral geneticdeterminants critical that are associated with the activity ofmaturation inhibitors map to amino acid residues flanking the HIV-1CA-SP1 cleavage site. When this determinant is introduced into theCA-SP1 cleavage sites of PA-457DSB-resistant non-HIV-1 viruses,maturation inhibitor sensitive chimeras result. These CA-SP1 chimericviruses serve as the basis for an animal efficacy model for HIV-1maturation inhibitors.

The region of HIV-1 CA-SP1 necessary for maturation inhibitorsensitivity is introduced into selected lentiviruses. Amino acidresidues from HIV-1 CA-SP1 junction that are determinants of PA-457DSBsensitivity were used to replace the corresponding CA-SP1 amino acids inthe genome of Simian Immunodeficiency (SIV). Similarly, the amino acidresidues from HIV-1 CA-SP1 junction that are determinants of PA-457DSBcan be replaced in Feline Immunodeficiency virus (FIV), BovineImmunodeficiency Virus (BIV), Equine Infectious Anemia Virus (EIAV),Visna-Maedi, and Caprine Arthritis Encephalitis virus (CAEV). Table 2depicts the Gag polypeptide sequence for HIV-1, SIV, FUV, EIAV and BIVin the region of the CA-SP1 cleavage site.

TABLE 2 Sequence comparison in the region of the CA-SP1 cleavage siteregion of HIV-1 with SIV, FIV, EIAV and BIV CA SP1 NC HIV-1 GHKARVLAEAMSQVTNPATIM IQKG (SEQ ID NO: 76) FIV GY K MQLL AE A LTKVQ VVQS (SEQID NO: 77) EIAV KQ K MMLL AK A LQ TGLA (SEQ ID NO: 78) BIV KS K MQFL VAA MKEMGIQSPIPAVLPHTPEAYA SQTS (SEQ ID NO: 79)

The HIV-1 CA-SP1 sequence used for replacement is as follows:

  CA       SP1 GHKARVL AEAMSQV (SEQ ID NO: 80)

The method described above for generating the SHIV CA-SP1 chimericprovirus DNA clone is used to generate FIV, EIAV and BIV provirus clonescontaining selected residues or extended region from CA-SP1 region ofHIV-1 replacing the corresponding wild-type sequence (FIG. 16).

The SHIV CA-SP1 chimeric-provirus DNA clone was generated bysite-directed mutagenesis employing standard molecular biologytechniques. Briefly, the unique restriction enzyme sites in the SIV Gagthat surrounding the CA-SP1 region were identified i.e., BamHI (inmatrix) and Sbf-I (in NC). Starting from the CA-SP1 region where themutagenisis is intended two overlapping primers, a forward and a reverseprimer incorporating the mutated sequence i.e., HIV CA-SP1 at their 5′ends were synthesized. Using the wild-type SIV provirus DNA as atemplate, two separate PCR reactions were set up to amplify SIV-Gagfragments in either direction from the site of mutagenisis (CA-SP1region), i.e., yield two amplified fragments that overlapped in themutated CA-SP1 region, a Bam HI-CA-SP1 fragment and a CA-SP1-Sbf-Ifragment. In a third PCR reaction, the fragments, Bam HI-CA-SP1 andCA-SP1-Sbf-I were annealed at their common HIV-CA-SP1 sequence andamplified with a forward SIV Bam HI primer and a reverse SIV Sbf-Iprimer to generate a full-length chimeric SHIV CA-SP1 gag fragment. Thechimeric SHIV CA-SP1 PCR fragment was cloned into BamHI-Sbf-I window ofSIV provirus clone replacing the SIV-Gag wild-type sequence to yield theSHIV CA-SP1 provirus cDNA clone.

Similarly, unique restriction enzyme cloning sites surrounding theCA-SP1 regions in FIV (Genbank Accession # NC_(—)001482), EIAV (GA#AF016316), and BIV (GA# M32690) genome have been identified (FIG. 16).Specific FIV/EIAV/BIV-HIV1CA-SP1 chimeric primers along with genomespecific primers of FIV, EIAV or BIV incorporating the specific cloningsite sequence are synthesized. These primers along with correspondingprovirus DNA clone as template (FIV, EIAV or BIAV) in PCR reactions togenerate the Chimeric HIV-1 CA-SP1 fragment. The chimeric HIV-1 CA-SP1fragment is digested with the appropriate restriction enzyme and clonedinto SacI-EcoRI window of FIV provirus; or (ii) KasI-EcoRV window ofEIAV provirus; or (iii) BsrGI-ApaI window of BIV provirus replacing thecorresponding wild type sequence (FIG. 16). The chimericFIV/EIAV/BIV-HIV-1CA-SP1 provirus DNA clones are sequenced to confirmthe presence of intended mutations. Based on observed results thatindicate the transfer of PA-457DSB sensitivity, additional constructsare generated employing the above strategy in order to optimize theresults.

In summary, a chimeric virus was generated in which the CA-SP1determinant of HIV-1 maturation inhibitor sensitivity has replaced theanalogous region of Gag in the maturation inhibitor-resistant simianimmunodeficiency virus (SIV). Transfer of this region of HIV-1 into thegenome of SIV results in a maturation inhibitor-sensitive phenotype.Infection of a non-human primate with this HIV-1/SIV chimeric virusshould result in an animal efficacy model for therapeutic development ofmaturation inhibitors.

Analogous approaches are used to prepare and characterize HIV-1 CA-SP1chimeras with FIV, BIV and EIAV. These additional DSB-sensitive chimericviruses should enable the development of additional animal efficacymodels for the study of HIV-1 maturation inhibitors.

Uses of Mutant and Chimeric Viruses

The mutant and chimeric viruses of the present invention, as describedabove, are useful in a variety of cell based as well as animal basedassays.

by comparing the phenotypes associated with a virus that is resistant toDSB, with a virus that is sensitive DSB, one may identify compounds thatact by a mechanism similar to that of DSB. Thus the invention includes amethod of identifying a compound that inhibits cleavage of p25 to p24 inwild type HIV-1, but does not inhibit CA-SP1 processing in HIV-1containing a deletion in the CA-SP1 region. Compounds obtained by such amethod are also included in the present invention.

Chimeras of SIV and other lentiviruses that do not readily infect humanshave additional advantages. Firstly, these viruses pose a lesser safetyhazard to laboratory workers. As a result, cell based assays to identifynovel compounds that inhibit CA-SP1 processing, for example, can beconducted with less risk. The lower risk may allow assays to beperformed that cannot be performed readily or safely with HIV, and mayalso lower the cost of such assays.

Furthermore, such chimeric viruses are useful in animal models. Forexample chimeric SIV that is sensitive to DSB may be used to identifynovel compounds that inhibit CA-SP1 processing, for example; to identifypharmaceutical compositions, routes of administration and dosage regimesfor treatment of disease; and for studying the effect of combinationtherapies, such as DSB with protease inhibitors.

As SIV is generally limited to infection of monkeys, the generation ofadditional lentiviral chimeras allows animal studies to be performed inanimals that are less expensive, easier to handle, have a faster diseaseprogression or otherwise more appropriate for a particular aspect ofhuman disease, for example.

Furthermore, animal models may be used to identify appropriatepharmaceutical compositions for the treatment of animal diseases, ofinterest in the treatment of companion animals and other high valueanimals, such as agricultural breeding stock and race horses.

Chimeric viruses may be derived from any retrovirus. For example,derived HIV-2, HTLV-I, HTLV-II, SIV, avian leukosis virus (ALV),endogenous avian retrovirus (EAV), mouse mammary tumor virus (MMTV),feline immunodeficiency virus (FIV), Bovine immunodeficiency virus(BIV), caprine arthritis encephalitis virus (CAEV), Equine infectiousanemia virus (EIAV), Visna-maedi virus, or feline leukemia virus (FeLV).

Such chimeric viruses may be used in the methods of the inventiondescribed elsewhere herein. For example, such recombinant non-HIV-1lentiviruses may be used in a method of identifying a compound whichinhibit processing of the viral Gag p25 protein (CA-SP1) to p24 (CA),the method consisting of comparing of the ability of said compound toinhibit replication of a wild-type non-HIV-1 lentivirus with theDSB-sensitive recombinant variant thereof. Such inhibition may occur ina cell; in an animal; or in vitro.

Construction and Use of Viruses or Polypeptides with Epitope Tags

The present invention is also drawn to recombinant retroviruses withepitope tags in the CA-SP1 region of Gag. Epitope tags may be insertedin the CA domain and/or in the SP1 domain. Suitable tags are well knownto those of ordinary skill in the art, and include haemagglutininepitope HA (YPYDVPDYA) (SEQ ID NO: 81), bluetongue virus epitope VP7(QYPALT) (SEQ ID NO: 82), α-tubulin epitope (EEF), Flag (DYKDDDDK) (SEQID NO: 83), and VSV-G (YTDOEMNRLGK) (SEQ ID NO: 84). Examples of SP1containing epitope tags are illustrated in FIG. 17.

Such epitope tagged viruses and fragments thereof are useful inidentifying novel compounds that inhibit CA-SP1 processing in vitro, incell based assays, and in vivo, including in animal models. Additionaluses of such epitope tagged viruses and fragments thereof are describedelsewhere herein.

Polynucleotides, Polypeptides and Antibodies of the Invention

The invention also includes isolated polypeptides and polynucleotides.In one embodiment, the invention includes polypeptides at least about40%, 50%, 60%, 70%, 80%, 90%, 99% or 100% identical to an amino acidsequence selected from the group consisting of: (a)KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPH KARILAEAMSQVTNSATIM (SEQ IDNO: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22); (c) TACQGVGGPSHKARILAEAMSQVTNSATIM(SEQ ID NO: 23); (d) MTACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 24);(e) SHKARILAEAMSQV (SEQ ID NO: 25); and (f) GHKARVLAEAMSQV (SEQ ID NO:26).

In another embodiment the invention includes polynucleotides encodingthe above polypeptides. Polynucleotides of the invention includedegenerate variants, such as those that differ in the third base of thecodon but nevertheless encodes the same amino acid due to coding“degeneracy”.

The term “about” as used herein refers to a value that is 10% more orless than the stated value, and preferably is 5% more or less.

The polypeptides and polynucleotides of the invention are useful in themethods of the invention. In one aspect, they may be used in an in vitroassay to identify compounds that bind to the CA-SP1 region of Gag. Inanother, they may be used in the production of antibodies useful inother methods described elsewhere herein. In another, a polynucleotidemay be inserted into a vector and thereupon into a host cell forproduction of polypeptide. The above embodiments are exemplary and arenot intended to be limiting.

The present invention comprises a polynucleotide comprising a sequencewhich encodes an amino acid sequence containing a mutation in the HIVGag p25 protein (CA-SP1), said mutation resulting in a decrease in theinhibition of processing of p25 (CA-SP1) to p24 (CA) by DSB. Thepolynucleotide of the invention includes a mutation which is optionallylocated at or near the CA-SP1 cleavage site or located in the SP1 domainof CA-SP1. Said mutation can be present in an amino acid sequence thatis selected from the group consisting of GHKARVLVEAMSQV (SEQ ID NO: 2)and SHKARILAEVMSQV (SEQ ID NO: 3). The polynucleotide of this inventionis also drawn to sequences designated as SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8 and SEQ ID NO: 9. The invention also includes a vectorcomprising said polynucleotide, a host cell comprising said vector and amethod of producing said polypeptides comprising incubating said hostcell in a medium and recovering the polypeptide from the medium.

The invention further includes a polynucleotide that hybridizes understringent conditions to a polynucleotide selected from the groupconsisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9.The invention also includes a polynucleotide which hybridizes to SEQ NO:5, SEQ ID NO: 7 or SEQ ID NO: 10 or 12, which contains a mutation whichresults in the decrease in the inhibition of processing of p25 to p24 by3-O-(3′,3′-dimethylsuccinyl)betulinic acid, and also wherein saidmutation is optionally located at or near the CA-SP1 cleavage site or inthe SP1 domain of CA-SP1. The invention is also directed to a vectorcomprising said polynucleotides, a host cell comprising said vector anda method of producing said polypeptides, comprising incubating said hostcell in a medium and recovering said polypeptide from the medium.

“Near” or “adjacent,” as used herein in reference to polypeptides ismeant to include about 50, about 25, about 20, or about 15 residues fromthe point of reference. For example, near may encompass about 50, about25, about 20 or about 15 residues on either side of the HIV-1 Gag CA-SP1cleavage site; more preferably about ten residues on either side of theHIV-1 Gag CA-SP1 cleavage site; and most preferably about seven residueson either side of the HIV-1 Gag CA-SP1 cleavage site. In reference topolynucleotides, the terms “near” or “adjacent refer to about 150, about75, about 60, about 45, or about 30 nucleotides from the point ofreference.

“Isolated” means altered “by the hand of man” from the natural state. Ifan “isolated” composition or substance occurs in nature, it has beenchanged or removed from its original environment, or both. Also,“isolated” nucleic acid molecule(s) of the invention is intended anucleic acid molecule, DNA or RNA, which has been removed from itsnative environment For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

“Polynucleotide” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritiated bases and unusual bases such as inosine.A variety of modifications has been made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

“Polypeptide” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. “Polypeptide” refers to both shortchains, commonly referred to as peptides, oligopeptides or oligomers,and to longer chains, generally referred to as proteins. Polypeptidesmay contain amino acids other than the 20 gene-encoded amino acids.“Polypeptides” include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from post-translation natural processesor may be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

“Mutant” as the term is used herein, is a polynucleotide or polypeptidethat differs from a reference polynucleotide or polypeptiderespectively. A typical mutant of a polynucleotide differs in nucleotidesequence from another, reference polynucleotide. Changes in thenucleotide sequence of the mutant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below. A typical mutant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A mutant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. A mutant of apolynucleotide or polypeptide may be a naturally occurring such as anallelic variant, or it may be a mutant that is not known to occurnaturally. Non-naturally occurring mutants of polynucleotides andpolypeptides may be made by mutagenesis techniques or by directsynthesis.

Thus, the mutant, (or fragments, derivatives or analogs) of apolypeptide encoded by any one of the polynucleotides described hereinmay be (i) one in which at least one or more of the amino acid residuesare substituted with a conserved or non-conserved amino acid residue (aconserved amino acid residue(s), or at least one but less than tenconserved amino acid residues) and such substituted amino acid residuemay or may not be one encoded by the genetic code, (ii) one in which oneor more of the amino acid residues includes a substituent group, (iii)one in which the mature polypeptide is fused with another compound, suchas a compound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as an IgG:Fc fusion regionpeptide or leader or secretory sequence or a sequence which is employedfor purification of the mature polypeptide or a proprotein sequence.Such mutants are deemed to be within the scope of those skilled in theart from the teachings herein. Polynucleotides encoding these mutantsare also encompassed by the invention. “Mutant” as used herein isequivalent to the term “variant.”

Substitutions of charged amino acids with another charged amino acidsand with neutral or negatively charged amino acids are included.Additionally, one or more of the amino acid residues of the polypeptidesof the invention (e.g., arginine and lysine residues) may be deleted orsubstituted with another residue to eliminate undesired processing byproteases such as, for example, furins or kexins. The prevention ofaggregation is highly desirable. Aggregation of proteins not onlyresults in a loss of activity but can also be problematic when preparingpharmaceutical formulations, because they can be immunogenic. (Pinckardet al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes36:838-845 (1987); Cleland et al Crit. Rev. Therapeutic Drug CarrierSystems 10:307-377 (1993)). Thus, the polypeptides of the presentinvention may include one or more amino acid substitutions, deletions oradditions, either from natural mutations or human manipulation.

As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table 3).

TABLE 3 Conservative Amino Acid Substitutions Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

However, in some embodiments, it is desirable to use nonconservativesubstitutions of amino acids. For example nonconservative substitutionof amino acids is used to render a DSB sensitive virus resistant to DSB.

The polynucleotides encompassed by this invention may have at leastabout 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%identity with a reference sequence, providing the referencepolynucleotide encodes an amino acid sequence containing a mutation inthe CA-SP1 protein, said mutation which results in the decrease in theinhibition of processing of p25 to p24 by a3-O-(3′,3′-dimethylsuccinyl)betulinic acid. The polynucleotides alsoencompassed by this invention include those mutations which are“silent,” in which different codons encode the same amino acid (wobble).

“Identity” is a measure of the identity of nucleotide sequences or aminoacid sequences. The term “identity” is used interchangeably with theword “homology” herein. In general, the sequences are aligned so thatthe highest order match is obtained. “Identity” per se has anart-recognized meaning and can be calculated using published techniques.While there exist a number of methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans. Methods commonly employed to determineidentity or similarity between two sequences include, but are notlimited to, those disclosed in Baxevanis and Oullette, Bioinformatics: APractical Guide to the Analysis of Genes and Proteins, Second Edition,Wiley-Interscience, New York, (2001). Methods to determine identity andsimilarity are codified in computer programs. Preferred computer programmethods to determine identity and similarity between two sequencesinclude, but are not limited to, GCS program package (Devereux, J. etal., Nucleic Acids Research 12(1):387, (1984)), BLASTP, BLASTN, FASTA(Atschul, S. F. et al., J. Molec. Biol. 215:403, (1990)).

A polynucleotide having a nucleotide sequence having at least, forexample, 95% “identity” to a reference nucleotide sequence is intendedthat the nucleotide sequence of the polynucleotide is identical to thereference sequence except that the polynucleotide sequence may includeup to five point mutations per each 100 nucleotides of the referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. These mutations ofthe reference sequence may occur at the 5′ or 3′ terminal positions ofthe reference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

Similarly, by a polypeptide having an amino acid sequence having atleast, for example, 95% “identity” to a reference amino acid sequence,is intended that the amino acid sequence of the polypeptide is identicalto the reference sequence except that the polypeptide sequence mayinclude up to five amino acid alterations per each 100 amino acids ofthe reference amino acid. To obtain a polypeptide having an amino acidsequence at least 95% identical to a reference amino acid sequence, upto 5% of the amino acid residues in the reference sequence may bedeleted or substituted with another amino acid, or a number of aminoacids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence. The reference (query) sequence may be the entirenucleotide sequence of any one of the nucleotide sequences of theinvention or any polynucleotide fragment (e.g., a polynucleotideencoding the amino acid sequence of the invention and/or C terminaldeletion).

Whether any particular nucleic acid molecule having at least 40%, 50%,60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% identity or whichare identical to, for instance, the nucleotide sequences of theinvention can be determined conventionally using known computer programssuch as the BESTFIT program (Wisconsin Sequence Analysis Package,Version 8 for Unix, Genetics Computer Group, University Research Park,575 Science Drive, Madison, Wis. 53711). BESTFIT uses the local homologyalgorithm of Smith and Waterman, (Advances in Applied Mathematics2:482-489 (1981)), to find the best segment of homology between twosequences. When using BESTFIT or any other sequence alignment program todetermine whether a particular sequence is, for instance, 95% identicalto a reference sequence according to the present invention, theparameters are set, such that the percentage of identity is calculatedover the full length of the reference nucleotide sequence and that gapsin homology of up to 5% of the total number of nucleotides in thereference sequence are allowed.

In a specific embodiment, the identity between a sequence of the presentinvention and a subject sequence, also referred to as a global sequencealignment, is determined using the FASTDB computer program based on thealgorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).Preferred parameters used in a FASTDB alignment of DNA sequences tocalculate percent identity are: Matrix=Unitary, k-tuple=4, MismatchPenalty=1, Joining Penalty=30, Randomization Group Length=0, CutoffScore=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or thelength of the subject nucleotide sequence, whichever is shorter.According to this embodiment, if the subject sequence is shorter thanthe reference sequence because of 5′ or 3′ deletions, not because ofinternal deletions, a manual correction is made to the results to takeinto consideration the fact that the FASTDB program does not account for5′ and 3′ truncations of the subject sequence when calculating percentidentity. For subject sequences truncated at the 5′ or 3′ ends, relativeto the query sequence, the percent identity is corrected by calculatingthe number of bases of the query sequence that are 5′ and 3′ of thesubject sequence, which are not matched/aligned, as a percent of thetotal bases of the query sequence. A determination of whether anucleotide is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This correctedscore is what is used for the purposes of this embodiment. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score. For example, a 90 base subject sequence is aligned to a100 base query sequence to determine percent identity. The deletionsoccur at the 5′ end of the subject sequence and therefore, the FASTDBalignment does not show a matched/alignment of the first 10 bases at 5′end. The 10 unpaired bases represent 10% of the sequence (number ofbases at the 5′ and 3′ ends not matched/total number of bases in thequery sequence) so 10% is subtracted from the percent identity scorecalculated by the FASTDB program. If the remaining 90 bases wereperfectly matched the final percent identity would be 90%. In anotherexample, a 90 base subject sequence is compared with a 100 base querysequence. This time the deletions are internal deletions so that thereare no bases on the 5′ or 3′ of the subject sequence, which are notmatched/aligned with the query. In this case the percent identitycalculated by FASTDB is not manually corrected. Only bases 5′ and 3′ ofthe subject sequence which are not matched/aligned with the querysequence are manually corrected. No other manual corrections are madefor the purposes of this embodiment.

The present application is directed to nucleic acid molecules having atleast about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%or 99% identity or which is identical to the nucleic acid sequencedisclosed herein, or fragments thereof, irrespective of whether theyencode a polypeptide having the disclosed functional activity. This isbecause even where a particular nucleic acid molecule does not encode apolypeptide having the disclosed functional activity, one of skill inthe art would still know how to use the nucleic acid molecule, forinstance, as a hybridization probe or a polymerase chain reaction (PCR)primer. Uses of the nucleic acid molecules of the present invention thatdo not encode a polypeptide having the disclosed functional activityinclude, inter alia: (1) isolating the variants thereof in a cDNAlibrary; (2) in situ hybridization (e.g., “FISH”) to determine cellularlocation or presence of the disclosed sequences, and (3) Northern Blotanalysis for detecting mRNA expression in specific tissues.

As used herein, the term “PCR” refers to the polymerase chain reactionthat is the subject of U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mulliset al., as well as improvements now known in the art. In accordance withthe present invention there may be employed conventional molecularbiology, microbiology, and recombinant DNA techniques within the skillof the art. Such techniques are explained fully in the literature. See,for example, Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: ALaboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

The term “stringent conditions,” as used herein refers to homology inhybridization, is based upon combined conditions of salt, temperature,organic solvents, and other parameters typically controlled inhybridization reactions, and well known in the art (Sambrook, et al.supra). The invention includes an isolated nucleic acid moleculecomprising, a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule of the invention described above, for instance, thesequence complementary to the coding and/or noncoding (i.e.,transcribed, untranslated) sequence of any polynucleotide or apolynucleotide fragment as described herein. By “stringent hybridizationconditions” is intended overnight incubation at 42° C. in a solutioncomprising, or alternatively consisting of: 50% formamide, 5×SSC (750 mMNaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA, followed by washing in 0.1×SSC at about 65° C.Polypeptides encoded by these polynucleotides are also encompassed bythe invention.

The invention also includes a virus comprising the polynucleotides ofthe invention, and wherein the virus includes a retrovirus comprisingsaid polynucleotides, and wherein the retrovirus may be a member of thegroup consisting of HIV-1, HIV-2, HTLV-I, HTLV-II, SIV, avian leukosisvirus (ALV), endogenous avian retrovirus (EAV), mouse mammary tumorvirus (MMTV), feline immunodeficiency virus (FIV), or feline leukemiavirus (FeLV).

The invention further includes a polypeptide containing a mutation inthe CA-SP1 protein, said mutation which results in the decrease ininhibition of processing of p25 to p24 by3-O-(3′,3′-dimethylsuccinyl)betulinic acid, and also wherein saidmutation is optionally located at or near the CA-SP1 cleavage site orlocated in the SP1 domain of SEQ ID NO: 5 or SEQ ID NO: 7 (parentalpolynucleotide sequences) encoding the CA-SP1 protein. Said polypeptidemay be encoded by a polynucleotide selected from the group consisting ofSEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9, or maycomprise a sequence that is selected from the group consisting ofGHKARVLVEAMSQV (SEQ ID NO: 2) and SHKARILAEVMSQV (SEQ ID NO: 3). Thepolypeptide of this invention may further be encoded by a polynucleotidewhich hybridizes to a polynucleotide selected from the group consistingof SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9. Theinvention also includes a polypeptide encoded by a polynucleotide whichhybridizes to SEQ NO: 5, SEQ ID NO: 7 or SEQ ID NO: 10 or 12, whichcontains a mutation that results in decrease in inhibition of processingof p25 to p24 by 3-O-(3′,3′-dimethylsuccinyl)betulinic acid, and alsowherein said mutation is optionally located at or near the CA-SP1cleavage site or in the SP1 domain of CA-SP1. The polypeptide of thisinvention further includes polypeptides that are part of a chimeric orfusion protein. Said chimeric proteins may be derived from species whichinclude, but are not limited to: primates, including simian and human;rodentia, including rat and mouse; feline; bovine; ovine; including goatand sheep; canine; or porcine. Fusion proteins may include syntheticpeptide sequences, bifunctional antibodies, peptides linked withproteins from the above species, or with linker peptides. Polypeptidesof the invention may be further linked with detectable labels; metalcompounds; cofactors; chromatography separation tags, such as, but notlimited to: histidine, protein A, or the like, or linkers; bloodstabilization moieties such as, but not limited to: transferrin, or thelike; therapeutic agents, and so forth.

The invention also includes an antibody which selectively binds an aminoacid sequence containing a mutation in the CA-SP1 protein that resultsin a decrease in the inhibition of processing of p25 (CA-SP1) to p24(CA) by 3-O-(3′,3′-dimethylsuccinyl)betulinic acid and also wherein saidmutation is optionally located at or near the CA-SP1 cleavage site or inthe SP1 domain of CA-SP1. The invention also includes an antibody whichselectively binds the polypeptide having a mutation which comprises asequence that is one of GHKARVLVEAMSQV (SEQ ID NO: 2), SHKARILAEVMSQV(SEQ ID NO: 3). Said antibody can selectively bind the polypeptideencoded by a polynucleotide sequence selected from the group consistingof SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9. Saidantibody can also selectively bind the polypeptide encoded by apolynucleotide which hybridizes under highly stringent conditions to apolynucleotide selected from the group consisting of SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9. The invention also includes anantibody that selectively binds SP1, which would enable one todistinguish SP1 from CA-SP1 (p25). The invention also includes anantibody that selectively binds CA (p24), which would enable one todistinguish CA from CA-SP1. The invention also includes an antibody thatselectively binds CA-SP1, which would enable one to distinguish CA fromCA-SP1. The invention additionally includes an antibody that selectivelybinds at or near the CA-SP1 cleavage site. The antibody of thisinvention may be a polyclonal antibody, a monoclonal antibody or saidantibody may be chimeric or bifunctional, or part of a fusion protein.The invention further includes a portion of any antibody of thisinvention, including single chain, light chain, heavy chain, CDR,F(ab′)₂, Fab, Fab′, Fv, sFv, or dsFv, or any combinations thereof.

As used herein, an antibody “selectively binds” a target peptide when itbinds the target peptide and does not significantly bind to unrelatedproteins. The term “selectively binds” also comprises determiningwhether the antibody selectively binds to the target mutant sequencerelative to a native target sequence. An antibody which “selectivelybinds” a target peptide is equivalent to an antibody which is “specific”to a target peptide, as used herein. An antibody is still considered toselectively bind a peptide even if it also binds to other proteins thatare not substantially homologous with the target peptide so long as suchproteins share homology with a fragment or domain of the peptide targetof the antibody. In this case, it would be understood that antibodybinding to the peptide is still selective despite some degree ofcross-reactivity. In another embodiment, the determination whether theantibody selectively binds to the mutant target sequence comprises: (a)determining the binding affinity of the antibody for the mutant targetsequence and for the native target sequences; and (b) comparing thebinding affinities so determined, the presence of a higher bindingaffinity for the mutant target sequence than for the native indicatingthat the antibody selectively binds to the mutant target sequence.

The invention is further drawn to an antibody immobilized on aninsoluble carrier comprising any of the antibodies disclosed herein. Theantibody immobilized on an insoluble carrier includes multiple wellplates, culture plates, culture tubes, test tubes, beads, spheres,filters, electrophoresis material, microscope slides, membranes, oraffinity chromatography medium.

The invention also includes labeled antibodies, comprising a detectablesignal. The labeled antibodies of this invention are labeled with adetectable molecule, which includes an enzyme, a fluorescent substance,a chemiluminescent substance, horseradish peroxidase, alkalinephosphatase, biotin, avidin, an electron dense substance, and aradioisotope, or any combination thereof.

The invention further includes a method of producing a hybridomacomprising fusing a mammalian myeloma cell with a mammalian B cell thatproduces a monoclonal antibody which selectively binds an amino acidsequence containing a mutation in the CA-SP1 protein, said mutationresulting in a decrease in the inhibition of processing of p25 to p24 by3-O-(3′,3′-dimethylsuccinyl)betulinic acid and a hybridoma producing anyof the monoclonal antibodies disclosed herein. The invention furtherincludes a method of producing an antibody comprising growing ahybridoma producing the monoclonal antibodies disclosed herein in anappropriate medium and isolating the antibodies from the medium, as iswell known in the art. The invention also includes the production ofpolyclonal antibodies comprising the injection, either one injection ormultiple injections of any of the polypeptides of the inventions intoany animal known in the art to be useful for the production ofpolyclonal antibodies, including, but not limited to mouse, rat,hamster, rabbit, goat, sheep, deer, guinea pig, or primate, andrecovering the antibodies in sera produced therein. The inventionincludes high avidity or high affinity antibodies produced therein. Theinvention also includes B cells produced from the listed species to befurther used in cell fusion procedures for the manufacture of monoclonalantibody-producing hybridomas as disclosed herein.

The invention is further drawn to a kit comprising the antibody or aportion thereof as disclosed herein, a container comprising saidantibody and instructions for use, a kit comprising the polypeptides ofthis invention and instructions for use and a kit comprising thepolynucleotide of the invention, a container comprising saidpolynucleotide and instructions for use, or any combinations thereof.These kits would include, but not be limited to nucleic acid detectionkits, which may, or may not, utilize PCR and immunoassay kits. Such kitsare useful for clinical diagnostic use and provide standardized reagentsas required in current clinical practice. These kits could eitherprovide information as to the presence or absence of mutations prior totreatment or monitor the progress of the patient during therapy. Thekits of the invention may also be used to provide standardized reagentsfor use in research laboratory studies.

Compounds of the Invention

In one aspect, the invention is also directed to a compound, a method ofusing a compound, a method of identifying a compound and the like.

The term “a”, “an” or “one”, as used in the present invention may referto either the singular or the plural. For example, “a compound”encompasses one or more compounds.

Compounds useful in the methods of the present invention includederivatives of betulinic acid and betulin that are presented in U.S.Pat. Nos. 5,679,828 and 6,172,110 respectively, and in U.S. applicationNos. 60/443,180 and 10/670,797, which are herein incorporated byreference. Additional useful compounds include oleanolic acidderivatives disclosed by Zhu et al. (Bioorg. Chem. Lett. 11:3115-3118(2001)); oleanolic acid and pomolic acid derivatives disclosed byKashiwada et al. (J. Nat. Prod. 61:1090-1095 (1998)); 3-O-acyl ursolicacid derivatives described by Kashiwada et al. (J. Nat. Prod.63:1619-1622 (2000)); and 3-alkylamido-3-deoxy-betulinic acidderivatives, disclosed by Kashiwada et al. (Chem. Pharm. Bull.48:1387-1390 (2000)). (All references incorporated by reference).

Compounds useful in the present invention include, but are not limitedto those betulinic acid derivatives having the general Formula I anddihydrobetulinic acid derivatives of Formula II:

or a pharmaceutically acceptable salt thereof, wherein,

R is a C₂-C₂₀ substituted or unsubstituted carboxyacyl,

R′ is hydrogen or a C₂-C₁₀ substituted and unsubstituted alkyl or arylgroup. Preferred compounds are those wherein R is one of thesubstituents in Table 4, below, and R′ is hydrogen.

Additional useful compounds include derivatives of betulin anddihydrobetulin of Formula III:

or a pharmaceutically acceptable salt thereof, wherein,

R₁ is a C₂-C₂₀ substituted or unsubstituted carboxyacyl,

R₂ is hydrogen, C(C₆H₅)₃, or a C₂-C₂₀ substituted or unsubstitutedcarboxyacyl; and

R₃ is hydrogen, halogen, amino, optionally substituted mono- ordi-alkylamino, or —OR₄, where R₄ is hydrogen, C₁₋₄ alkanoyl, benzoyl, orC₂-C₂₀ substituted or unsubstituted carboxyacyl;

wherein the dashed line represents an optional double bond between C20and C29.

Preferred compounds useful in the present invention are those where

R₁ is one of the substituents in Table 4, R₂ is hydrogen or one of thesubstituents in Table 4 and R₃ is hydrogen.

TABLE 4 Preferred Substituents for R₁, R₂.

More preferred compounds are 3-O-(3′,3′-dimethylsuccinyl)betulinic acid,3-O-(3′,3′-dimethylsuccinyl)dihydrobetulinic acid,3-O-(3′,3′-dimethylsuccinyl)betulin, and3-O-(3′,3′-dimethylsuccinylglutaryl)dihydrobetulin.

A particularly preferred compound is3-O-(3′,3′-dimethylsuccinyl)betulinic acid.

Additional compounds useful in the present invention are described bythe Formulas IV, V, VI and VII.

-   R₁₁=—OR₁₄ or —NHR₁₅;-   R₁₂=COOR₁₇, COO⁻A⁺, or CHOR₁₇-   R₁₃=—H, halogen, amino, optionally substituted mono- or    di-alkylamino, or —OR₁₆;-   R₁₄—H, C₂-C₂₀ substituted or unsubstituted carboxyacyl;-   R₁₅=—H, C₂-C₂₀ substituted or unsubstituted carboxyacyl;-   R₁₆=—H, C₄-C₇ alkanoyl, benzyloyl, or C₂-C₂₀ substituted or    unsubstituted carboxyacyl;-   R₁₇=—H, C(C₆H₅)₃, or C₂-C₂₀ substituted or unsubstituted    carboxyacyl;

wherein dashed line represents optional bond between C₂₀ and C₂₉, and

wherein A=Na+, K⁺, or other cation

VII

R₃₁ R₃₂ R₃₃ R₃₄ R₃₅ R₃₆ R₃₇ R₃₈ R₃₉ R₄₀ R₄₁ 1 CH₂NH₂ H H H H H H H H H H2

H H H H H H

H H H 3

H H H H H H

H H H 4 COOH H H H H H H H H H H 5 COOH H H H H H H

H H H 6 COOH H H H H H H

H H H 7 COOH H H H H H H

H H H 8 COOH H H H H H H

H H H 9 COOH H H H H H H

H H H 10 COOH H H H H H H

H H H 11 COOH H H H H H H

H H H 12 COOH H H H H H H

H H H 13 COOH H H H H H H

H H H 14 COOH H H H H H H

H H H 15 COOH H H H H H H

H H H 16 COOH H H H H H H H H H H 17 COOH H H H H H OH H H OH H 18 COOHH H H H H OH H OH H H 19 COOH H H H H H H H H H OH 20 COOH H OH H H H HH H H H 21 COOH H H H H H H H H H H 22 COOH H H H H H OH H H H H 23 COOHH H H H H OH H OH H H 24 COOH H OH H H H OH H H H H 25 COOH H OH H H O HH H H 26 COOH H OH H OH O H H H H 27 COOH H OH H OH OH H H H H H 28 COOHH OH H OH H OH H H H H 29 COOH H OH H HH OH H H H H 30 COOH CH₃ H H H HH H H H H 31 COOH CH₃ H H H H H

H H H 32 COOH H H CH₃ H H H H H H H 33 COOH H H H H H H

H H H

R₃₈ moieties other than hydrogen are attached to R₃₃ oxygen by acovalent bond to the carbonyl carbon.

Preferred compounds are those where R38 is not hydrogen.

Compounds useful in the methods of the invention also include thosedescribed in U.S. Provisional Application No. 60/559,358, which isentirely incorporated by reference. In one aspect, these compounds aredescribed by reference to the following compounds VIII to XI:

Additional compounds useful in the present invention have the generalFormula VIII:

or a pharmaceutically acceptable salt or ester thereof:

wherein A is a fused ring of formula

wherein the ring carbons designated x and y in the formulas of A are thesame as the ring carbons designated x and y in Formula VIII;

R₁ is a carboxyalkanoyl, where the alkanoyl chain can be optionallysubstituted by one or more hydroxy or halo, or can be interrupted by anitrogen, sulfur or oxygen atom, or combinations thereof;

R₂, R₃ and R₄ are independently hydrogen, methyl, halogen, or hydroxy;

R₅ is carboxyalkoxycarbonyl, alkoxycarbonyl, alkanoyloxymethyl,carboxyalkanoyloxymethyl, alkoxymethyl or carboxyalkoxymethyl, any ofwhich is optionally substituted by one or more hydroxy or halo, or R₅ isa carboxyl or hydroxymethyl;

R₆ is hydrogen, methyl, hydroxy or halogen;

R₇ and R₈ are independently hydrogen or C₁₋₆ alkyl;

R₉ is CH₂ or CH₃;

R₁₀ is hydrogen, hydroxy or methyl;

R₁₁ is methyl, methoxycarbonyl, carboxyalkoxycarbonyl,alkanoyloxymethyl, alkoxymethyl or carboxyalkoxymethyl, any of which isoptionally substituted by one or more hydroxy or halo;

R₁₂ is hydrogen or methyl;

R₁₃ is hydrogen or methyl;

R₁₄ is hydrogen or hydroxy;

R₁₅ is hydrogen if C12 and C13 form a single bond, or R₁₅ is absent ifC12 and C13 form a double bond; and

wherein the straight dashed line represents an optional double bondbetween C12 and C13 or C20 and C29;

with the proviso that when A is

then R₁ cannot be glutaryl or succinyl when a double bond exists betweenC12 and C13;

when A is (ii) and R₁₁ is methyl, then R₁ cannot be succinyl;

when A is (iii) and R₂, R₃ and R₁₃ are each hydrogen, then R₁ cannot besuccinyl; and

with the proviso that A (i) cannot be

when R₂ and R₃ are both methyl and a double bond exists between C12 andC13.

In some embodiments, R₁ is a carboxy(C₂₋₆)alkylcarbonyl group or acarboxy(C₂₋₆)alkoxy(C₁₋₆)alkylcarbonyl group. Suitable groups areselected from the group consisting of:

According to the invention, in some embodiments the compounds haveFormula IX:

wherein R₁, R₄, R₅, R₆, R₇, R₈ and R₁₄ are as defined above for FormulaVIII. In one embodiment, R₆ is β-methyl, R₈ is hydrogen, R₅ ishydroxymethyl and R₁ is 3′,3′-dimethylglutaryl, 3′,3′-dimethylsuccinyl,glutaryl or succinyl. In another embodiment, R₆ is hydrogen, R₇ and R₈are both methyl, R₅ is carboxyl and R₁ is 3′,3′-dimethylglutaryl,3′,3′-dimethylsuccinyl, glutaryl or succinyl.

In some embodiments, R₅ is carboxyalkoxycarbonyl, alkoxycarbonyl,alkanoyloxymethyl, carboxyalkanoyloxymethyl, alkoxymethyl orcarboxyalkoxymethyl, any of which is optionally substituted by one ormore hydroxy or halo, or R₅ is a carboxyl or hydroxymethyl. In someembodiments, R₅ is selected from a group consisting of carboxyl,hydroxymethyl, —CO₂(CH₂)_(n)COOH, —CO₂(CH₂)_(n)CH₃,—CH₂OC(O)(CH₂)_(n)CH₃, —CH₂OC(O)(CH₂)_(n)COOH, —CH₂—O—(CH₂)_(n)CH₃ and—CH₂—O—(CH₂)_(n)COOH. In some embodiments, R₅ is selected from a groupconsisting of

In some embodiments, R₅ is hydroxymethyl. In some embodiments, R₅ iscarboxyl. In some embodiments, n is from 0 to 20. In some embodiments, nis from 1 to 10. In some embodiments, n is from 2 to 8. In someembodiments, n is from 1 to 6. In some embodiments, n is from 2 to 6.

In some embodiments, compounds useful in the present invention have theFormula X:

wherein R₁, R₉, R₁₀, and R₁₁ are as defined above for Formula VIII. Inone embodiment, R₁ is 3′,3′-dimethylglutaryl, 3′,3′-dimethylsuccinyl,glutaryl or succinyl.

In some embodiments, R₁₁ is methyl, methoxycarbonyl,carboxyalkoxycarbonyl, alkanoyloxymethyl, alkoxymethyl orcarboxyalkoxymethyl, any of which is optionally substituted by one ormore hydroxy or halo. In some embodiments, R₁₁ is selected from thegroup consisting of methyl, —CO₂(CH₂)_(n)COOH, —CH₂OC(O)(CH₂)_(n)CH₃,—CH₂—O—(CH₂)_(n)CH₃ and —CH₂—O—(CH₂)_(n)COOH.

In some embodiments, n is from 0 to 20. In some embodiments, n is from 1to 10. In some embodiments, n is from 2 to 8. In some embodiments, n isfrom 1 to 6. In some embodiments, n is from 2 to 6. In some embodiments,R₁₁ is methyl. In some embodiments, R₁₁ is methoxycarbonyl. In someembodiments, R₁₁ is selected from the group consisting of methoxymethyland ethoxymethyl. In some embodiments, methyl groups found in R₁₁ can besubstituted with a halogen or a hydroxy.

In some embodiments, the compounds useful in the present invention haveFormula XI:

wherein R₁, R₂, R₃, R₄, and R₁₃ are as defined above for Formula VIII.In one embodiment, R₁ is 3′,3′-dimethylglutaryl, 3′,3′-dimethylsuccinyl,glutaryl or succinyl. In one embodiment, both R₂ and R₃ are methyl.

Any triterpene which falls within the scope of Formula VIII can be used.According to the invention, in some embodiments the compounds of FormulaVIII are selected from the group consisting of derivatives of uvaol,ursolic acid, erythrodiol, echinocystic acid, oleanolic acid,sumaresinolic acid, lupeol, dihydrolupeol, betulinic acid methylester,dihydrobetulinic acid methylester, 17-α-methyl-androstanediol,androstanediol, and 4,4-dimethyl-androstanediol.

In some embodiments, the compounds of the present invention are definedas in Formula VIII, wherein R₂ and R₃ are both methyl. In someembodiments, the compounds of the present invention are defined as inFormula VIII, wherein R₁ is 3′,3′-dimethylsuccinyl. In some embodiments,the compounds of the present invention are defined as in Formula VIII,wherein R₁ is succinyl, i.e.,

According to the invention, in some embodiments the stereochemistry ofthe sidechain substituents is important. In some embodiments, thecompounds of the present invention are defined as in Formula VIII,wherein A is (i) and R₅ is in the β position. In some embodiments, thecompounds of the present invention are defined as in Formula VIII,wherein A is (i) and R₆ is in the β position. In some embodiments, thecompounds of the present invention are defined as in Formula VIII,wherein A is (i) and R₁₄ is in the α position. In some embodiments, thecompounds of the present invention are defined as in Formula VIII,wherein A is (i), R₇ is α-methyl, and R₈ is hydrogen. In someembodiments, the compounds of the present invention are defined as inFormula VIII, wherein A is (i), R₈ is α-methyl, and R₇ is hydrogen. Insome embodiments, the compounds of the present invention are defined asin Formula VIII, wherein A is (i) and both R₇ and R₈ are methyl. In someembodiments, the compounds of the present invention are defined as inFormula VIII, wherein A is (ii) and R₁₁ is in the β position.

In some embodiments, 3′,3′-dimethylsuccinyl is at the C3 position. Insome embodiments, the compounds of Formula IX are3-O-(3′,3′-dimethylsuccinyl)uvaol;3-O-(3′,3′-dimethylsuccinyl)erythrodiol;3-O-(3′,3′-dimethylsuccinyl)echinocystic acid or3-O-(3′,3′-dimethylsuccinyl)sumaresinolic acid. In some embodiments, thecompounds of Formula X are 3-O-(3′,3′-dimethylsuccinyl)lupeol;3-O-(3′,3′-dimethylsuccinyl)dihydrolupeol;3-O-(3′,3′-dimethylsuccinyl)17β-methylester-betulinic acid; or3-O-(3′,3′-dimethylsuccinyl) 17β-methylester-dihydrobetulinic acid. Insome embodiments, the compounds of Formula XI are3-O-(3′,3′-dimethylsuccinyl) 4,4-dimethylandrostanediol;3-O-(3′,3′-dimethylsuccinyl) 17α-methylandrostanediol;3-O-(3′,3′-dimethylsuccinyl)androstanediol.

Alkyl groups and alkyl containing groups of the compounds of the presentinvention can be straight chain or branched alkyl groups, preferablyhaving one to ten carbon atoms. In some embodiments, the alkyl groups oralkyl containing groups of the present invention can be substituted witha C₃₋₇ cycloalkyl group. In some embodiments, the cycloalkyl group mayinclude, but is not limited to, a cyclobutyl, cyclopentyl or cyclohexylgroup.

Also, included within the scope of the present invention are thenon-toxic pharmaceutically acceptable salts of the compounds of thepresent invention. These salts can be prepared in situ during the finalisolation and purification of the compounds or by separately reactingthe purified compound in its free acid form with a suitable organic orinorganic base and isolating the salt thus formed. These can includecations based on the alkali and alkaline earth metals, such as sodium,lithium, potassium, calcium, magnesium, and the like, as well asnontoxic ammonium, quaternary ammonium and amine cations including, butnot limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, ethylamine, N-methylglucamine and the like.

Also, included within the scope of the present invention are thenon-toxic pharmaceutically acceptable esters of the compounds of thepresent invention. Ester groups are preferably of the type which arerelatively readily hydrolyzed under physiological conditions. Examplesof pharmaceutically acceptable esters of the compounds of the inventioninclude C₁₋₆ alkyl esters wherein the alkyl group is a straight orbranched chain. Acceptable esters also include C₅₋₇ cycloalkyl esters aswell as arylalkyl esters, such as, but not limited to benzyl. C₁₋₄ alkylesters are preferred. In some embodiments, the esters are selected fromthe group consisting of alkylcarboxylic acid esters, such as acetic acidesters, and mono- or dialkylphosphate esters, such as methylphoshateester or dimethylphosphate ester. Esters of the compounds of the presentinvention can be prepared according to conventional methods.

Certain compounds within the scope of Formulae VIII, IX, X and XI arederivatives referred to as “prodrugs”. The expression “prodrug” refersto compounds that are rapidly transformed in vivo by an enzymatic orchemical process, to yield the parent compound of the above formulas,for example, by hydrolysis in blood. A thorough discussion is providedby Higuchi, T. and V. Stella in Pro-drugs as Novel Delivery Systems,Vol. 14, A.C.S. Symposium Series, and in Bioreversible Carriers in DrugDesign, Ed. Edward B. Roche, American Pharmaceutical Association,Pergamon Press, 1987. Useful prodrugs can be esters of the compounds ofFormulae VIII, IX, X, and XI. In some prodrug embodiments, a lower alkylgroup is substituted with one or more hydroxy or halo groups by asuitable acid. Suitable acids include, e.g., carboxylic acids, sulfonicacids, phosphoric acid or lower alkyl esters thereof, and phosphonicacid or lower alkyl esters thereof. For example, suitable carboxylicacids include alkylcarboxylic acids, such as acetic acid, arylcarboxylicacids and arylalkylcarboxylic acids. Suitable sulfonic acids includealkylsulfonic acids, arylsulfonic acids and arylalkylsulfonic acids.Suitable phosphoric and phosphonic acid esters are methyl or ethylesters. In some embodiments, the C3 acyl groups having dimethyl groupsor oxygen at the C3′ position can be the most active compounds. Thisobservation suggests that these types of acyl groups might be importantto the enhanced anti-HIV activity.

In an additional embodiment, the invention includes compounds andmethods that use compounds of Formula XII:

where R₇₂ is one of:

wherein

X is hydroxy or halogen; and

R₇₃ is lower alkyl, such as methyl, ethyl or propyl.

In one embodiment, the invention is drawn to a method of treating HIV-1infection in a patient by administering a compound that inhibitsprocessing of the viral Gag p25 protein (CA-SP1) to p24 (CA), but doesnot significantly affect other Gag processing steps, wherein saidcompound is a compound of Formula I through XII

In one embodiment, the invention is drawn to a method of treating HIV-1infection in a patient by administering a compound that inhibitsprocessing of the viral Gag p25 protein (CA-SP1) to p24 (CA), but doesnot significantly affect other Gag processing steps, wherein saidcompound is a compound of Formula I through XII, with the exception ofDSB.

In one embodiment, the invention is drawn to a method of treating HIV-1infection in a patient by administering a compound that inhibitsprocessing of the viral Gag p25 protein (CA-SP1) to p24 (CA), but doesnot significantly affect other Gag processing steps, wherein saidcompound is other than a compound of Formula I through VII. In oneembodiment, the invention is drawn to a method of treating HIV-1infection in a patient by administering a compound that inhibitsprocessing of the viral Gag p25 protein (CA-SP1) to p24 (CA), but doesnot significantly affect other Gag processing steps, wherein saidcompound is other than a compound of Formula I through XI.

In another embodiment, the invention is drawn to a method of inhibitingprocessing of the viral Gag p25 protein (CA-SP1) to p24 (CA) in a cell,but without significantly affect other Gag processing steps, whereinsaid compound is a compound of Formula Groups I through XII.

In another embodiment, the invention is drawn to a method of inhibitingprocessing of the viral Gag p25 protein (CA-SP1) to p24 (CA) in a cell,but without significantly affect other Gag processing steps, whereinsaid compound is other than a compound of Formula I through VII.

In another embodiment, the invention is drawn to a method of inhibitingprocessing of the viral Gag p25 protein (CA-SP1) to p24 (CA) in a cell,but without significantly affect other Gag processing steps, whereinsaid compound is other than a compound of Formula I through XI.

In another embodiment, the invention is drawn to a method of treatinghuman blood or blood products by inhibiting processing of the viral Gagp25 protein (CA-SP1) to p24 (CA) in a cell, but without significantlyaffect other Gag processing steps, wherein said compound is a compoundof Formula I through XII.

In another embodiment, the invention is drawn to a method of treatinghuman blood or blood products by inhibiting processing of the viral Gagp25 protein (CA-SP1) to p24 (CA) in a cell, but without significantlyaffect other Gag processing steps, wherein said compound is other than acompound of Formula I through VII.

In another embodiment, the invention is drawn to a method of treatinghuman blood or blood products by inhibiting processing of the viral Gagp25 protein (CA-SP1) to p24 (CA) in a cell, but without significantlyaffect other Gag processing steps, wherein said compound is other than acompound of Formula I through XI.

Synthesis of DSB and Related Compounds

Reaction of betulinic acid and dihydrobetulinic acid withdimethylsuccinic anhydride produced a mixture of3-O-(2′,2′-dimethylsuccinyl) and 3-O-(3′,3′-dimethylsuccinyl)-betulinicacid and dihydrobetulinic acid, respectively. The mixtures weresuccessfully separated by preparative scale HPLC yielding pure samples.The structures of these isomers were assigned by long-range ¹H-¹³C COSYexaminations.

The derivatives of betulinic acid and dihydrobetulinic acid of thepresent invention were all synthesized by refluxing a solution ofbetulinic acid or dihydrobetulinic acid, dimethylaminopyridine (1equivalent mol), and an appropriate anhydride (2.5-10 equivalent mol) inanhydrous pyridine (5-10 mL). The reaction mixture was then diluted withice water and extracted with CHCl₃. The organic layer was washed withwater, dried over MgSO₄, and concentrated under reduced pressure. Theresidue was chromatographed using silica gel column orsemi-preparative-scale HPLC to yield the product.

Preparation of 3-O-(3′,3′-dimethylsuccinyl)betulinic acid: yield 70%(starting with 542 mg of betulinic acid); crystallization from MeOH gavecolorless needles; mp 274°-276° C.; [α]_(D) ¹⁹+23.5° (c=0.71),CHCl₃-MeOH [1:1]); Positive FABMS m/z 585 (M+H)⁺; Negative FABMS m/z 583(M−H)⁻; HR-FABMS calcd for C₃₆H₅₇O₆ 585.4155, found m/z 585.4161; ¹H NMR(pyridine-d₅): 0.73, 0.92, 0.97, 1.01, 1.05 (each 3H, s; 4-(CH₃)₂,8-CH₃, 10-CH₃, 14-CH₃), 1.55 (6H, s, 3′-CH₃×2), 1.80 (3H, s, 20-CH₃),2.89, 2.97 (each 1H, d, J=15.5 Hz, H-2′), 3.53 (1H, m, H-19), 4.76 (1H,dd, J=5.0, 11.5 Hz, H-3), 4.78, 4.95 (each 1H, br s, H-30).

3-O-(3′,3′-dimethylsuccinyl)dihydrobetulinic acid: yield 24.5% (startingwith 155.9 mg of dihydrobetulinic acid); crystallization from MeOH—H₂Ogave colorless needles; mp 291°-292° C.; [α]_(D) ²⁰−13.4° (c=1.1,CHCl₃-MeOH [1:1], ¹H NMR (pyridine-d₅): 0.85, 0.94 (each 3H, d, J=7.0Hz; 20-(CH₃)₂), 0.75, 0.93, 0.97, 1.01, 1.03 (each 3H, s; 4-(CH₃)₂,8-CH₃, 10-CH₃, 14-CH₃), 1.55 (6H, s; 3′-CH₃×2), 2.89, 2.97 (each 1H, d,J=15.5 Hz; H-2′), 4.77 (1H, dd, J=5.0, 11.0 Hz, H-3); Anal. Calcd forC₃₆H₅₈O₆.5/2H₂O: C, 68.43; H, 10.04; found C, 68.64; H 9.78.

The synthesis of 3-O-(3′,3′-dimethylglutaryl)betulinic acid wasdisclosed U.S. Pat. No. 5,679,828, as COMPOUND NO. 4.

3-O-(3′,3′-dimethylglutaryl)dihydrobetulinic acid: yield 93.3% (startingwith 100.5 mg of dihydrobetulinic acid); crystallization from needlesMeOH—H₂O gave colorless needles; mp 287°-289° C.; [α]_(D) ²⁰−17.9°(c=0.5, CHCl₃-MeOH[1:1]); ¹H-NMR (pyridine-d₅): 0.86, 0.93 (each 3H, d,J=6.5 Hz; 20-(CH₃)₂), 0.78, 0.92, 0.96, 1.02, 1.05 (each 3H, s;4-(CH₃)₂, 8-CH₃, 10-CH₃, 14-CH₃), 1.38, 1.39 (each 3H, s; 3′-CH₃×2),2.78 (4H, m, H₂-2′ and 4′), 4.76 (1H, dd, J=4.5, 11.5 Hz; H-3). Anal.Calcd for C₃₇H₆₀O₆: C, 73.96; H, 10.06; found C, 73.83; H 10.10.

The synthesis for 3-O-diglycolyl-betulinic acid was disclosed in U.S.Pat. No. 5,679,828, as COMPOUND NO. 5.

3-O-diglycolyl-dihydrobetulinic acid: yield 79.2% (starting with 103.5mg of dihydrobetulinic acid); an off-white amorphous powder; [α]_(D)²⁰−9.8° (c=1.1, CHCl₃-MeOH[1:1]); ¹H-NMR (pyridine-d₅): 0.79, 0.87 (each3H, d, J=6.5 Hz; 20-(CH₃)₂), 0.87, 0.88, 0.91, 0.98, 1.01 (each 3H, s;4-(CH₃)₂, 8-CH₃, 10-CH₃, 14-CH₃), 4.21, 4.23 (each 2H, s, H₂-2′ and 4′),4.57 (1H, dd, J=6.5, 10.0 Hz, H-3); Anal. Calcd for C₃₄H₅₄O₇.2H₂O: C,66.85; H, 9.57; found C, 67.21; H, 9.33.

The syntheses of 3-O-(3′,3′-dimethylsuccinyl)betulin and3-O-(3′,3′-dimethylglutaryl)betulin were disclosed in U.S. applicationSer. No. 10/670,797.

Methods of Inhibiting HIV with a Compound

Methods of “inhibiting HIV” or “inhibition of HIV” as used herein, meansany interference in, inhibition of, and/or prevention of HIV using themethods of the invention. As such, methods of inhibition are useful ininhibiting with the infectivity of HIV, inhibition of p25 processing,inhibition of viral maturation, formation of virions that exhibitaltered phenotypes, and the like. Preferably, methods of the inventionact upon p25 processing in the cells of an animal, but are not limitedby that method of action.

A method of inhibiting HIV with a compound may be relevant to a methodof treating HIV infection in a patient. Therefore, a method ofinhibiting HIV with a compound may similarly be used to treat a patient.

The methods of inhibiting HIV-1 replication in cells of an animalincludes contacting infected cells with a compound of Formula I throughXII, above. Related embodiments include a method of treating a HIV-1infectionin a patient comprising administration of a compound of FormulaI through XII; a method of inhibiting p25 processing either in a cell,in vivo, and/or in vitro, by administration of a compound that inhibitssaid p25 processing; and a method of treating human blood or bloodproducts by administering a compound of Formula I through XII. Alsoincluded are a method of identifying a compound that inhibits any one ofp25 processing, HIV maturation, HIV infectivity, HIV virion phenotypesand the like.

In one embodiment, the compound is a derivative of betulinic acid,betulin, or dihydrobetulinic acid or dihydrobetulin and which includesthe preferred substituents of Table 4. Preferred compounds include butare not limited to 3-O-(3′,3′-dimethylsuccinyl)betulinic acid,3-O-(3′,3′-dimethylsuccinyl)betulin,3-O-(3′,3′-dimethylglutaryl)betulin, 3-O-(3′,3′-dimethylsuccinyl)dihydrobetulinic acid,3-O-(3′,3′-dimethylglutaryl)betulinic acid,(3′,3′-dimethylglutaryl)dihydrobetulinic acid, 3-O-diglycolyl-betulinicacid, and 3-O-diglycolyl-dihydrobetulinic acid.

In one embodiment, the invention is drawn to a method inhibiting HIV-1replication in cells of an animal by contacting infected cells with acompound that inhibits processing of the viral Gag p25 protein (CA-SP1)to p24 (CA), but does not significantly affect other Gag processingsteps, wherein said compound is a compound of Formulas I through XIIabove.

In one embodiment, the invention is drawn to a method of inhibitingHIV-1 replication in cells of an animal by contacting infected cellswith a compound that inhibits processing of the viral Gag p25 protein(CA-SP1) to p24 (CA), but does not significantly affect other Gagprocessing steps, wherein said compound is compound of Formulas Ithrough XII, with the exception of DSB.

In one embodiment, the invention is drawn to a method of inhibitingHIV-1 replication in cells of an animal by contacting infected cellswith a compound that inhibits processing of the viral Gag p25 protein(CA-SP1) to p24 (CA), but does not significantly affect other Gagprocessing steps, wherein said compound is one that is excluded fromthose of Formulas I through VI. In one embodiment, the invention isdrawn to a method of treating HIV-1 infection in a patient byadministering a compound that inhibits processing of the viral Gag p25protein (CA-SP1) to p24 (CA), but does not significantly affect otherGag processing steps, wherein said compound is one that is other thanthose of Formulas I through XI.

In another embodiment, the invention is drawn to a method of inhibitingprocessing of the viral Gag p25 protein (CA-SP1) to p24 (CA) in a cell,but without significantly affecting other Gag processing steps, whereinsaid compound is a compound of Formulas I through XII.

In another embodiment, the invention is drawn to a method of inhibitingprocessing of the viral Gag p25 protein (CA-SP1) to p24 (CA) in a cell,but without significantly affecting other Gag processing steps, whereinsaid compound is a compound other than those of Formulas I through VI.

In another embodiment, the invention is drawn to a method of inhibitingprocessing of the viral Gag p25 protein (CA-SP1) to p24 (CA) in a cell,but without significantly affecting other Gag processing steps, whereinsaid compound is a compound other than those of Formulas I through XI.

In another embodiment, the invention is drawn to a method of inhibitingprocessing of the viral Gag p25 protein (CA-SP1) to p24 (CA) in a cell,but without significantly affecting other Gag processing steps, whereinsaid compound is a compound other than those of Formulas I through XI.In another embodiment, the invention is drawn to a method of treatinghuman blood or blood products by inhibiting processing of the viral Gagp25 protein (CA-SP1) to p24 (CA) in a cell, but without significantlyaffect other Gag processing steps, wherein said compound is a compoundof Formulas I through XII.

In another embodiment, the invention is drawn to a method of treatinghuman blood or blood products by inhibiting processing of the viral Gagp25 protein (CA-SP1) to p24 (CA) in a cell, but without significantlyaffect other Gag processing steps, wherein said compound is a compoundother than those of Formulas I through VI.

In another embodiment, the invention is drawn to a method of treatinghuman blood or blood products by inhibiting processing of the viral Gagp25 protein (CA-SP1) to p24 (CA) in a cell, but without significantlyaffect other Gag processing steps, wherein said compound is a compoundother than those of Formulas I through XI.

The method disclosed herein, further comprises contacting said cellswith one or more drugs selected from the group consisting of anti-viralagents, anti-fungal agents, anti-bacterial agents, anti-cancer agents,immunostimulating agents, and combinations thereof. The method mayinclude the treatment of human blood products.

The invention may also be used in conjunction with a method of treatingcancer comprising the administration to an animal of one or moreanti-neoplastic agents, exposing an animal to a cancer cell-killingamount of radiation, or a combination of both.

Methods of Identifying Compounds

The invention further includes a method for identifying compounds thatinhibit HIV-1 replication in cells of an animal disclosed herein. In oneembodiment, said method comprises:

(a) contacting a Gag polypeptide comprising a CA-SP1 cleavage site witha test compound;

(b) adding a labeled substance that selectively binds at or near theCA-SP1 cleavage site; and

(c) measuring the binding of the test compound at or near the CA-SP1cleavage site.

Labeled substances or molecules include labeled antibodies or labeledDSB and the label includes an enzyme, fluorescent substance,chemiluminescent substance, horseradish peroxidase, alkalinephosphatase, biotin, avidin, electron dense substance, such as gold,osmium tetroxide, lead or uranyl acetate, and radioisotope, antibodieslabeled with such substances of molecules or a combination thereof. Theassays could include, but are not limited to ELISA, single and doublesandwich techniques, immunodiffusion or immunoprecipitation techniques,as known in the art (“Immunoassay Handbook, 2^(nd) ed.,” D. Wild, NaturePublishing Group, (2001)). Said methods of identifying also couldinclude, but are not limited to Western blot assays, calorimetricassays, light and electron microscopic techniques, confocal microscopy,or other techniques known in the art.

A method of identifying compounds that inhibit HIV replication in cellsof an animal further comprises:

(a) contacting a Gag protein comprising a wild-type CA-SP1 cleavagesite, with HIV-1 protease in the presence of a test compound;

(b) separately, contacting a Gag protein comprising a mutant CA-SP1cleavage site or a protein comprising an alternative protease cleavagesite with HIV-1 protease in the presence of the test compound; and

(c) comparing the cleavage of the native wild-type Gag protein to theamount of cleavage of the mutant Gag protein or to the amount ofcleavage of the peptide comprising an alternative protease cleavagesite.

Step (b) above is performed as a control in order to eliminate compoundsthat might bind directly to, and therefore inhibit, the protease enzyme.The above method also includes the method wherein the wild-type CA-SP1,mutant CA-SP1 or alternative protease cleavage site is contained withina polypeptide fragment or recombinant peptide.

The method for identifying compounds that inhibit HIV-1 disclosedherein, also includes a method wherein said peptide or protein islabeled with a fluorescent moiety and a fluorescence quenching moiety,each bound to opposite sides of the CA-SP1 cleavage site, and whereinsaid detecting comprises measuring the signal from the fluorescentmoiety, or wherein said peptide or protein is labeled with twofluorescent moieties, each bound to opposite sides of the CA-SP1cleavage site, and wherein said detecting comprises measuring thetransfer of fluorescent energy from one moiety to the other in thepresence of the test compound and HIV-1 protease and comparing saidtransfer of fluorescent energy to that observed when the same procedureis applied to a peptide that comprises a sequence containing a mutationin the CA-SP1 cleavage site or that comprises a sequence containinganother cleavage site. Examples of fluorescence-based assays of proteaseactivity are well known in the art. In one such example, a proteasesubstrate is labeled with green fluorescent dye molecules, whichfluoresce when the substrate is cleaved by the protease enzyme(Molecular Probes, Protease Assay Kit).

The method of comparing the cleavage, above, also includes using alabeled antibody that selectively binds CA or SP1 or CA-SP1 in order tomeasure the extent to which the test compound inhibits CA-SP1 cleavage.The antibody can be labeled with a molecule selected from the groupconsisting of enzyme, fluorescent substance, chemiluminescent substance,horseradish peroxidase, alkaline phosphatase, biotin, avidin, electrondense substance, and radioisotope, or combinations thereof. The methodalso includes the use of an antibody to a specific epitope tag sequenceto selectively detect CA-SP1 (p25) or SP1, into which the amino acidsequence for that epitope tag has been engineered according to standardmethods in the art. Suitable tags are well known to those of ordinaryskill in the art, and include haemagglutinin epitope HA (YPYDVPDYA) (SEQID NO: 81), bluetongue virus epitope VP7 (QYPALT) (SEQ ID NO: 82),α-tubulin epitope (EEF), Flag (DYKDDDDK) (SEQ ID NO: 83), and VSV-G(YTDOEMNRLGK) (SEQ ID NO: 84). Examples of SP1 containing epitope tagsare illustrated in FIG. 17.

As an example, the sequence of the FLAG epitope tag (Sigma-Aldrich) isinserted into the p2 (SP1) region of Gag by oligonucleotide-directedmutagenesis of a Gag expression plasmid. The presence of the SP1 domainin the cell-expressed protein is then be detected using commerciallyavailable anti-FLAG monoclonal antibodies (Sigma-Aldrich). (Hopp, T. P.Biotechnology 6: 1204-1210 (1988)).

The method of identifying compounds that disrupt CA-SP1 cleavage alsoincludes the addition of a compound to cells infected with HIV-1 and thedetection of CA-SP1 cleavage products by lysing and analyzing the cellsor the released virions. The method included in the invention can beperformed using a western blot analysis of viral proteins and detectingp25 using an antibody to p25 or wherein said mixture is analyzed byperforming a gel electrophoresis of viral proteins and imaging ofmetabolically labeled proteins, or wherein the mixture is analyzed usingimmunoassays that use an antibody that selectively binds p25 or anantibody that selectively binds in order to distinguish p25 from p24.The invention includes the use of an antibody to a specific epitope tagsequence inserted into the C-terminal domain of SP1 to selectivelydetect p25 or SP1. For example, a sandwich ELISA assay can be performedwhere p25 and p24 in detergent-solubilized virus are captured using anantibody that selectively binds to the CA region of Gag, which antibodyis bound to a multiple well plate. Following a washing step, bound p25is detected using an antibody to an epitope tag inserted in SP1, whichis conjugated to an appropriate detection reagent (e.g. alkalinephosphatase for an enzyme-linked immunosorbent assay). Virus released bycells treated with compounds that act via this mechanism will generallyhave increased levels of p25 compared with untreated virions.

The disclosed method is drawn to an antibody that selectively binds p25,or an antibody that selectively binds SP1, or an antibody to an epitopetag sequence inserted into SP1, which is labeled with a moleculeselected from the group consisting of enzyme, fluorescent substance,chemiluminescent substance, horseradish peroxidase, alkalinephosphatase, biotin, avidin, electron dense substance, and radioisotope,or combinations thereof.

“Infected cells,” as used herein, includes cells infected naturally bymembrane fusion and subsequent insertion of the viral genome into thecells, or transfection of the cells with viral genetic material throughartificial means. These methods include, but are not limited to, calciumphosphate transfection, DEAE-dextran mediated transfection,microinjection, lipid-mediated transfection, electroporation orinfection.

The invention may be practiced by infecting target cells in vitro withan infectious strain of HIV and in the presence of test compound, underappropriate culture conditions and for varying periods of time. Infectedcells or supernatant fluid can be processed and loaded onto apolyacrylamide gel for the detection of virus levels, by methods thatare well known in the art. Non-infected and non-treated cells can beused as negative and positive infection controls, respectively.Alternatively, the invention may be practiced by culturing the targetcells in the presence of test compound prior to infecting the cells withan HIV strain.

The invention also includes a method for identifying compounds thatinhibit HIV-1 replication in the cells of an animal, comprising:

(a) contacting a test compound with wild-type virus isolates andseparately with virus isolates having reduced sensitivity to3-O-(3′,3′-dimethylsuccinyl)betulinic acid; and

(b) selecting test compounds that are more active against the wild-typevirus isolate compared with virus isolates that have reduced sensitivityto 3-O-(3′,3′-dimethylsuccinyl)betulinic acid.

This invention further includes a method for identifying compounds thatact by any of the abovementioned mechanism, involving treating HIV-1infected or transfected cells with a compound then analyzing the virusparticles released by compound-treated cells by thin-sectioning andtransmission electron microscopy, by standard methods well known in theart. A compound acts by the abovementioned mechanism if particles aredetected that exhibit spherical condensed cores that are acentric withrespect to the viral particle and/or a crescent-shaped electron-denselayer just inside the viral membrane.

For electron microscopic studies, infected cells or centrifuged viruspellets obtained from the supernatant fluid can be contacted with afixative, such as glutaraldehyde or freshly-made paraformaldehyde,and/or osmium tetroxide or other electron microscopy compatible fixativethat is known in the art. The virus from the supernatant fluid or thecells, is dehydrated and embedded in an electron-lucent polymer such asan epoxy resin or methacrylate, thin sectioned using an ultramicrotome,stained using electron dense stains such as uranyl acetate, and/or leadcitrate, and viewed in a transmission electron microscope. Non-infectedand non-treated cells can be used as negative and positive infectioncontrols, respectively. Alternatively, the invention may be practiced byculturing the target cells in the presence of test compound prior toinfecting the cells with an HIV strain. Maturation defects caused by thecompounds of the present invention are determined by the presence ofmorphologically aberrant viral particles, compared with controls, asdescribed herein.

For cell culture studies, the virus-infected cells may be observed forthe formation of syncytia, or the supernatant may be tested for thepresence of HIV particles. Virus present in the supernatant may beharvested to infect other naïve cultures to determine infectivity.

Also included in the invention, is a method of determining if anindividual is infected with HIV-1, is susceptible to treatment by acompound that inhibits p25 processing, the method involves taking bloodfrom the patient, genotyping the viral RNA and determining whether theviral RNA contains mutations in the CA-SP1 cleavage site.

The invention also includes a method for identifying compounds that actby the abovementioned mechanisms, involving testing by a combination ofthe methods disclosed herein.

HIV Gag protein and fragments thereof for use in the aforementionedassays may be expressed or synthesized using a variety of methodsfamiliar to those skilled in the art. Gag can be produced in an in vitrotranscription and translation system using a rabbit reticulocyte lysate.Gag expressed in this system has been shown to be processed sequentiallyin a pattern similar to that observed in infected cells (Pettit, S. C.et al. J. Virol. 76:10226-10233 (2002)). Moreover, Gag expressed by thismethod is capable of assembling into immature viral particles when fusedto a heterologous type D retroviral cytoplasmic self-assembly domain(Sakalian, M. et al., J. Virol. 76:10811-10820 (2002)). The plasmidpDAB72, available from the NIH AIDS Reagent Program can be used for thispurpose (Erickson-Viitanen, S. et al., AIDS Res. Hum. Retroviruses.5:577-91 (1989); Sidhu M. K. et al., Biotechniques, 18:20, 22, 24(1995)). Other in vitro transcription/translation systems based on wheatgerm or bacterial lysates can also be used for this purpose. HIV Gag mayalso be expressed in transfected cells using a variety of commerciallyavailable expression vectors. The plasmid p55-GAG/GFP, available fromthe NIH AIDS Reagent Program, may be used to express an HIV Gag-greenfluorescent protein fusion protein in mammalian cells for druginteraction studies (Sandefur, S. et al., J. Virol. 72:2723-2732(1998)). This construct would permit the capture and purification of Gagfusion protein using GFP-specific monoclonal antibodies. In addition,Gag may be expressed in cells using recombinant viral vectors, such asthose used in the vaccinia virus, adenovirus, or baculovirus systems.Gag can also be expressed by infecting cells with HIV or by transfectingcells with proviral DNA. Finally, Gag may be expressed in yeast orbacterial cells transformed with the appropriate expression vectors.

In addition to Gag proteins expressed in cells or in vitro using celllysates, peptides corresponding to various regions of Gag may becommercially synthesized from using standard peptide synthesistechniques.

The invention further encompasses compounds identified by the method ofthis invention and/or a compound which inhibits HIV-1 replicationaccording to the methods of this invention and pharmaceuticalcompositions comprising one or more compounds as disclosed herein, orpharmaceutically acceptable salts, esters or prodrugs thereof, andpharmaceutically acceptable carriers.

Pharmaceutical Compositions

Compounds according to the present invention have been found to possessanti-retroviral, particularly anti-HIV, activity. The salts and otherformulations of the present invention are expected to have improvedwater solubility, and enhanced oral bioavailability. Also, due to theimproved water solubility, it will be easier to formulate the salts ofthe present invention into pharmaceutical preparations. Further,compounds according to the present invention are expected to haveimproved biodistribution properties.

In one embodiment, the compounds are those of Formula I through XII, inanother they are compounds other than the compounds of Formula I throughXI.

This invention also includes a pharmaceutical composition comprising acompound that inhibits processing of the viral Gag p25 protein (CA-SP1)to p24 (CA), but has no significant effect on other Gag processingsteps, or that inhibits the maturation of virus particles released fromtreated infected cells, such as the compounds of Formula I through XII.The invention includes a pharmaceutical composition comprising one ormore compounds disclosed herein, or pharmaceutically acceptable salts,esters or prodrugs thereof, and pharmaceutically acceptable carriers,wherein said compound is of Formulae I through XII above, or preferably,wherein said compound is selected from the group consisting of3-O-(3′,3′-dimethylsuccinyl)betulinic acid,3-O-(3′,3′-dimethylsuccinyl)betulin,3-O-(3′,3′-dimethylglutaryl)betulin,3-O-(3′,3′-dimethylsuccinyl)dihydrobetulinic acid,3-O-(3′,3′-dimethylglutaryl)betulinic acid,(3′,3′-dimethylglutaryl)dihydrobetulinic acid, 3-O-diglycolyl-betulinicacid, and 3-O-diglycolyl-dihydrobetulinic acid. The pharmaceuticalcompositions according to the invention, further comprise one or moredrugs selected from an anti-viral agent, anti-fungal agent, anti-canceragent or an immunostimulating agent.

Pharmaceutical compositions of the present invention can comprise atleast one of the compounds of Formulae I through XII disclosed herein.Pharmaceutical compositions according to the present invention can alsofurther comprise other anti-viral agents such as, but not limited to,AZT (zidovudine, RETROVIR®, GlaxoSmithKline), 3TC (lamivudine, EPIVIR®,GlaxoSmithKline), AZT+3TC, (COMBIVIR®, GlaxoSmithKline),AZT+3TC+abacavir (TRIZIVIR®, GlaxoSmithKline), ddI (didanosine, VIDEX®,Bristol-Myers Squibb), ddC (zalcitabine, HIVID®, Hoffmann-La Roche), D4T(stavudine, ZERIT®, Bristol-Myers Squibb), abacavir (ZIAGEN®,GlaxoSmithKline), tenofovir (VIREAD®, Gilead Sciences), nevirapine(VIRAMUNE®, Boehringer Ingelheim), delavirdine (Pfizer), emtricitabine(EMTRIVA®, Gilead Sciences), efavirenz (SUSTIVA®, DuPontPharmaceuticals), saquinavir (INVIRASE®, FORTOVASE®, Hoffmann-LaRoche),ritonavir (NORVIR®, Abbott Laboratories), indinavir (CRIXIVAN®, Merckand Company), nelfinavir (VIRACEPT®, Pfizer), amprenavir (AGENERASE®,GlaxoSmithKline), adefovir (PREVEON®, HEPSERA®, Gilead Sciences),atazanavir (Bristol-Myers Squibb), fosamprenavir (LEXIVA®,GlaxoSmithKline) and hydroxyurea (HYDREA®, Bristol-Meyers Squibb), orany other antiretroviral drugs or antibodies in combination with eachother, or associated with a biologically based therapeutic, such as, forexample, gp41-derived peptides enfuvirtide (FUZEON®, Roche and Trimeris)and T-1249, or soluble CD4, antibodies to CD4, and conjugates of CD4 oranti-CD4, or as additionally presented herein.

Additional suitable antiviral agents for optimal use with one of thecompounds of Formulae I through XII of the present invention caninclude, but are not limited to, amphotericin B (FUNGIZONE®); Ampligen(mismatched RNA) developed by Hemispherx Biopharma; BETASERON®(β-interferon, Chiron); butylated hydroxytoluene; Carrosyn(polymannoacetate); Castanospermine; Contracan (stearic acidderivative); Creme Pharmatex (containing benzalkonium chloride);5-unsubstituted derivative of zidovudine; penciclovir (DENAVIR®Novartis); famciclovir (FAMVIR® Novartis); acyclovir (ZOVIRAX®GlaxoSmithKline); cytofovir (VISTIDE® Gilead); ganciclovir (CYTOVENE®,Hoffman LaRoche); dextran sulfate; D-penicillamine(3-mercapto-D-valine); FOSCARNET® (trisodium phosphonoformate;AstraZeneca); fusidic acid; glycyrrhizin (a constituent of licoriceroot); HPA-23 (ammonium-21-tungsto-9-antimonate); ORNIDYL®(eflornithine; Aventis); nonoxynol; pentamidine isethionate(PENTAM-300); Peptide T (octapeptide sequence; Peninsula Laboratories);Phenyloin (Pfizer); INH or isoniazid; ribavirin (VIRAZOLE®, ValeantPharmaceuticals); rifabutin, ansamycin (MYCOBUTIN® Pfizer); CD4-IgG2(Progenics Pharmaceuticals) or other CD4-containing or CD4-basedmolecules; Trimetrexate (Medimmune); suramin and analogues thereof(Bayer); and WELLFERON® (α-interferon, GlaxoSmithKline).

Pharmaceutical compositions of the present invention can also furthercomprise immunomodulators. Suitable immunomodulators for optional usewith a betulinic acid or betulin derivative of the present invention inaccordance with the present invention can include, but are not limitedto: ABPP (Bropririmine); Ampligen (mismatched RNA) Hemispherx Biopharma;anti-human interferon-α-antibody; ascorbic acid and derivatives thereof;interferon-β; Ciamexon; cyclosporin; cimetidine; CL-246,738; colonystimulating factors, including GM-CSF; dinitrochlorobenzene; HE2000(Hollis-Eden Pharmaceuticals); inteferon-γ; glucan; hyperimmunegamma-globulin (Bayer); immuthiol (sodium diethylthiocarbamate);interleukin-1 (Hoffmann-LaRoche; Amgen), interleukin-2 (IL-2) (Chiron);isoprinosine (inosine pranobex); Krestin; LC-9018 (Yakult); lentinan(Yamanouchi); LF-1695; methionine-enkephalin; Minophagen C; muramyltripeptide, MTP-PE; naltrexone (Barr Laboratories); RNA immunomodulator;REMUNE® (Immune Response Corporation); RETICULOSE® (Advanced ViralResearch Corporation); shosaikoto; ginseng; thymic humoral factor;Thymopentin; thymosin factor 5; thymosin 1 (ZADAXIN®, SciClone),thymostimulin, TNF (tumor necrosis factor, Genentech), and vitaminpreparations.

Pharmaceutical compositions of the present invention can also furthercomprise anti-cancer therapeutic agents. Suitable anti-cancertherapeutic agents for optional use include an anti-cancer compositioneffective to inhibit neoplasia comprising a compound, or apharmaceutically acceptable salt or prodrug of said anti-cancer agent,which can be used for combination therapy include, but are not limitedto alkylating agents, such as busulfan, cis-platin, mitomycin C, andcarboplatin antimitotic agents, such as colchicine, vinblastine, taxols,such as paclitaxel (TAXOL®, Bristol-Meyers Squibb) docetaxel (TAXOTERE®,Aventis), topo I inhibitors, such as camptothecin, irinotecan andtopotecan (HYCAMTIN®, GlaxoSmithKline), topo II inhibitors, such asdoxorubicin, daunorubicin and etoposides such as VP16; RNA/DNAantimetabolites, such as 5-azacytidine, 5-fluorouracil and methotrexate,DNA antimetabolites, such as 5-fluoro-2′-deoxy-uridine, ara-C,hydroxyurea, thioguanine, and antibodies, such as trastuzumab(HERCEPTIN®, Genentech), and rituximab (RITUXAN®, Genentech andBiogen-Idec), melphalan, chlorambucil, cyclophosamide, ifosfamide,vincristine, mitoguazone, epirubicin, aclarubicin, bleomycin,mitoxantrone, elliptinium, fludarabine, octreotide, retinoic acid,tamoxifen, alanosine, and combinations thereof.

The invention further provides methods for providing anti-bacterialtherapeutics, anti-parasitic therapeutics, and anti-fungal therapeutics,for use in combination with the compounds of the invention andpharmaceutically-acceptable salts thereof. Examples of anti-bacterialtherapeutics include compounds such as penicillins, ampicillin,amoxicillin, cyclacillin, epicillin, methicillin, nafcillin, oxacillin,cloxacillin, dicloxacillin, flucloxacillin, carbenicillin, cephalexin,cepharadine, cefadoxil, cefaclor, cefoxitin, cefotaxime, ceftizoxime,cefinenoxine, ceftriaxone, moxalactam, imipenem, clavulanate, timentin,sulbactam, erythromycin, neomycin, gentamycin, streptomycin,metronidazole, chloramphenicol, clindamycin, lincomycin, quinolones,rifampin, sulfonamides, bacitracin, polymyxin B, vancomycin,doxycycline, methacycline, minocycline, tetracycline, amphotericin B,cycloserine, ciprofloxacin, norfloxacin, isoniazid, ethambutol, andnalidixic acid, as well as derivatives and altered forms of each ofthese compounds.

Examples of anti-parasitic therapeutics include bithionol,diethylcarbamazine citrate, mebendazole, metrifonate, niclosamine,niridazole, oxamniquine and other quinine derivatives, piperazinecitrate, praziquantel, pyrantel pamoate and thiabendazole, as well asderivatives and altered forms of each of these compounds.

Examples of anti-fungal therapeutics include amphotericin B,clotrimazole, econazole nitrate, flucytosine, griseofulvin, ketoconazoleand miconazole, as well as derivatives and altered forms of each ofthese compounds. Anti-fungal compounds also include aculeacin A andpapulocandin B.

The preferred animal subject of the present invention is a mammal. Bythe term “mammal” is meant an individual belonging to the classMammalia. The invention is particularly useful in the treatment of humanpatients.

The term “treating” means the administering to subjects a compound ofFormulae I through XII or a compound identified by one or more assayswithin the present invention, for purposes which can include prevention,amelioration, or cure of a retroviral-related pathology. Said compoundsfor treating a subject that are identified by one or more assays withinthe present inventions are identified as compounds which have theability to disrupt Gag processing, described herein.

The term “inhibits the interaction” as used herein, means preventing, orreducing the rate of, direct or indirect association of one or moremolecules, peptides, proteins, enzymes, or receptors; or preventing orreducing the normal activity of one or more molecules, peptides,proteins, enzymes or receptors.

Medicaments are considered to be provided “in combination” with oneanother if they are provided to the patient concurrently or if the timebetween the administration of each medicament is such as to permit anoverlap of biological activity.

In one preferred embodiment, at least one compound of Formulae I throughXII above comprises a single pharmaceutical composition.

Pharmaceutical compositions for administration according to the presentinvention can comprise at least one compound of Formulae I through XIIabove or compounds identified by one or more assays within the presentinvention. Said compounds for treating a subject that are identified byone or more assays within the present inventions are identified ascompounds which have the ability to disrupt Gag processing, describedherein. The compounds according to the present invention are furtherincluded in a pharmaceutically acceptable form optionally combined witha pharmaceutically acceptable carrier. These compositions can beadministered by any means that achieve their intended purposes. Amountsand regimens for the administration of a compound of Formulae I throughXII according to the present invention can be determined readily bythose with ordinary skill in the clinical art of treating a retroviralpathology.

For example, administration can be by parenteral, such as subcutaneous,intravenous, intramuscular, intraperitoneal, transdermal, transmucosal,ocular, rectal, intravaginal, or buccal routes. Alternatively, orconcurrently, administration can be by the oral route. Theadministration may be as an oral or nasal spray, or topically, such aspowders, ointments, drops or a patch. The dosage administered dependsupon the age, health and weight of the recipient, type of previous orconcurrent treatment, if any, frequency of treatment, and the nature ofthe effect desired.

Compounds and methods of the invention are useful in additional ways.For example, such compounds may be used prophylatically, to minimize therisk of infection. In another embodiment, a compound may be used tominimize spread of the disease from an infected person.

The invention is also directed to novel methods of treating HIV in aninfected individual. In one embodiment, the invention is particularlyuseful in stimulating an immune response in a person infected with HIV.For example, by allowing noninfectious virus to be released frominfected cells, such infected cells continue to expose antigens and maybe effectively targeted by the immune system or other therapies directedagainst such antigens. In another example, by continuing to permit therelease of noninfectious virus, an infected individual continues todevelop an immune response to said virus without suffering thedeleterious effects of such a virus.

The invention is also useful in expanding the scope of treatment, andoffers novel means of treating disease in patients in need thereof. Inanother embodiment, the invention may be practiced in a patient who doesnot respond to other therapy for reasons other than viral resistance.For example, conventional methods of treating HIV, as known in the art,are associated with deleterious side effects. In one embodiment, themethods and compositions of the invention are useful in treating apatient without a reduction in one or more deleterious side effects. Inone embodiment the invention includes a method of treating a patientwith a compound that does not have a particular side effect or has lessof a particular side effect.

The bioavailability of drugs is also relevant in treatment. In anembodiment, the invention may be practiced such that compounds are moreeffectively absorbed into infected cells. In one embodiment theinvention encompasses improved methods of delivering a drug to a cellinfected with HIV.

Compositions within the scope of this invention include all compositionscomprising at least one compound of Formulae I through XII aboveaccording to the present invention in an amount effective to achieve itsintended purpose. While individual needs vary, determination of optimalranges of effective amounts of each component is within the skill of theart. For example, a dose may comprise 0.0001 mg to 10 g/kg of bodyweight. Typical dosages comprise about 0.1 to about 100 mg/kg bodyweight. The preferred dosages comprise about 1 to about 100 mg/kg bodyweight of the active ingredient. More preferred dosages comprise about 5to about 50 mg/kg body weight.

Administration of a compound of the present invention can alsooptionally include previous, concurrent, subsequent or adjunctivetherapy using immune system boosters or immunomodulators. In addition tothe pharmacologically active compounds, a pharmaceutical composition ofthe present invention can also contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically. Preferably, the preparations, particularlythose preparations which can be administered orally and which can beused for the preferred type of administration, such as tablets, dragees,and capsules, and also preparations which can be administered rectally,such as suppositories, as well as suitable solutions for administrationby injection or orally, contain from about 0.01 to 99 percent,preferably from about 20 to 75 percent of active compound(s), togetherwith the excipient.

Pharmaceutical preparations of the present invention are manufactured ina manner which is itself known, for example, by means of conventionalmixing, granulating, dragee-making, dissolving, or lyophilizingprocesses. Thus, pharmaceutical preparations for oral use can beobtained by combining the active compounds with solid excipients,optionally grinding the resulting mixture, and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

Suitable excipients are, e.g., fillers such as saccharide, for example,lactose or sucrose, mannitol or sorbitol; cellulose preparations and/orcalcium phosphates, such as tricalcium phosphate or calcium hydrogenphosphate; as well as binders such as starch paste, using, for example,maize starch, wheat starch, rice starch, potato starch, gelatin,tragacanth, cellulose, methyl cellulose, hydroxypropylmethylcellulose,sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,disintegrating agents can be added such as the above-mentioned starchesand also carboxymethyl starch, cross-linked polyvinyl pyrrolidone, agar,or alginic acid or a salt thereof, such as sodium alginate. Auxiliariesare, above all, flow-regulating agents and lubricants, for example,silica, talc, stearic acid or salts thereof, such as magnesium stearateor calcium stearate, and/or polyethylene glycol. Dragee cores areprovided with suitable coatings which, if desired, are resistant togastric juices. For this purpose, concentrated saccharide solutions canbe used, which can optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquersolutions and suitable organic solvents or solvent mixtures. In order toproduce coatings resistant to gastric juices, solutions of suitablecellulose preparations such as acetylcellulose phthalate orhydroxypropylmethyl cellulose phthalate are used. Dyestuffs or pigmentscan be added to the tablets or dragee coatings, for example, foridentification or in order to characterize combinations of activecompound doses.

Other pharmaceutical preparations which an be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules can contain the active compounds in the form of granules whichcan be mixed with fillers such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds are preferablydissolved or suspended in suitable liquids, such as fatty oils or liquidparaffin. In addition, stabilizers can be added.

Possible pharmaceutical preparations which can be used rectally include,for example, suppositories which consist of a combination of the activecompounds with a suppository base. Suitable suppository bases are, forexample, natural or synthetic triglycerides, or paraffin hydrocarbons.In addition, it is also possible to use gelatin rectal capsules whichconsist of a combination of the active compounds with a base. Possiblebase materials include, for example, liquid triglycerides, polyethyleneglycols, or paraffin hydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, for example,water-soluble salts. In addition, suspensions of the active compounds asappropriate oily injection suspensions can be administered. Suitablelipophilic solvents or vehicles include fatty oils, such as sesame oil,or synthetic fatty acid esters, such as ethyl oleate, triglycerides orglycol-400. Aqueous injection suspensions that can contain substanceswhich increase the viscosity of the suspension include, for example,sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally,the suspension can also contain stabilizers.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to the active compounds, the liquid dosage forms may containinert diluents commonly used in the art such as, for example, water orother solvents, solubilizing agents and emulsifiers such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethyl formamide, oils such as cottonseed, groundnut, corn,germ, olive, castor, and sesame oils, glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, cellulose, microcrystalline cellulose,aluminum metahydroxide, bentonite, agar-agar, and tragacanth, andcombinations thereof.

Pharmaceutical compositions for topical administration includeformulations appropriate for administration to the skin, mucosa,surfaces of the lung or eye. Compositions may be prepared as apressurized or non-pressurized dry powder, liquid or suspension. Theactive ingredients in non-pressurized powdered formulations may beadmixed in a finely divided form in a pharmaceutically-acceptable inertcarrier, including but not limited to mannitol, fructose, dextrose,sucrose, lactose, saccharin or other sugars or sweeteners.

The pressurized composition may contain a compressed gas, such asnitrogen, or a liquefied gas propellant. The propellant may also containa surface-active ingredient, which may be a liquid or solid non-ionic oranionic agent. The anionic agent may be in the form of a sodium salt.

A formulation for use in the eye would comprise a pharmaceuticallyacceptable ophthalmic carrier, such as an ointment, oils, such asvegetable oils, or an encapsulating material. The regions of the eye tobe treated include the corneal region, or internal regions such as theiris, lens, ciliary body, anterior chamber, posterior chamber, aqueoushumor, vitreous humor, choroid or retina.

Compositions for rectal administration may be in the form ofsuppositories. Compositions for use in the vagina may be in the form ofsuppositories, creams, foams, or in-dwelling vaginal inserts.

The compositions may be administered in the form of liposomes. Liposomesmay be made from phospholipids, phosphatidyl cholines (lecithins) orother lipoidal compounds, natural or synthetic, as known in the art. Anynon-toxic, pharmacologically acceptable lipid capable of formingliposomes may be used. The liposomes may be multilamellar ormono-lamellar.

A pharmaceutical formulation for systemic administration according tothe invention can be formulated for enteral, parenteral or topicaladministration. Indeed, all three types of formulation can be usedsimultaneously to achieve systemic administration of the activeingredient.

Suitable formulations for oral administration include hard or softgelatin capsules, dragees, pills, tablets, including coated tablets,elixirs, suspensions, syrups or inhalations and controlled release formsthereof.

The compounds of Formula I or II above or compounds identified by one ormore assays within the present invention and have the ability to disruptGag processing, can also be administered in the form of an implant whencompounded with a biodegradable slow-release carrier. Alternatively, thecompounds of the present invention can be formulated as a transdermalpatch for continuous release of the active ingredient.

The following examples are illustrative only and are not intended tolimit the scope of the invention as defined by the appended claims. Itwill be apparent to those skilled in the art that various modificationsand variations can be made in the methods of the present inventionwithout departing from the spirit and scope of the invention. Thus, itis intended that the present invention covers the modifications andvariations of this invention provided they come within the scope of theappended claims and their equivalents.

EXAMPLES Example 1 Anti-Viral Activity Against Primary HIV-1 Isolates

A robust virus inhibition assay was used to evaluate the anti-viralactivity of DSB against primary HIV-1 isolates propagated in PMBC.Briefly, serial dilutions of DSB were made in medium into 96-well tissueculture plates. 25-250 TCID₅₀ of virus and 5×10⁵ PHA-stimulated PBMCswere added to each well. On days 1, 3 and 5 post-infection, media wasremoved from each well and replaced with fresh media containing DSB atthe appropriate concentration. On day 7 post-infection, culturesupernatant was removed from each well for p24 detection of virusreplication and 50% inhibitory concentrations (IC₅₀) were calculated bystandard methods.

Table 5 shows the potent anti-viral activity of DSB against a panel ofprimary HIV-1 isolates. DSB exhibits levels of activity similar toapproved drugs that were tested in parallel. Importantly, the activityof DSB was not restricted by co-receptor usage.

TABLE 5 Inhibitory activity (IC₅₀) of DSB and two approved drugs againsta panel of primary Clade B HIV-1 isolates. Clinical HIV-1 isolatesdenoted by * were isolated at Panacos. All other virus isolates wereobtained from the NIH AIDS Reference Repository. IC₅₀ (nM) Virus Isolate# Co-Receptor usage DSB AZT Nevirapine BZ167 X4 4.0 2.2 31.2 92HT599 X49.8 5.8 25.3 US1 R5 5.6 0.9 22.1 19101N* R5 3.8 2.4 59.4 3401N* R5/X412.0 17.5 32.1 92US723 R5/X4 4.6 1.2 26.8 22101N* R5/X4 2.6 0.9 4.9 Mean6.1 4.4 28.8 Note: R5 and X4 refer to the chemokine receptors CCR5 andCXCR4 respectively.

Toxicity of DSB was analyzed by incubating with PHA-stimulated PBMC for7 days at a range of concentrations, then determining cell viabilityusing the XTT method. The 50% cytotoxic concentration was >30 μM,corresponding to an in vitro therapeutic index of approximately 5000.

Example 2 Anti-Viral Activity of DSB Against Drug Resistant HIV-1Isolates

The activity of DSB was tested against a panel of HIV-1 isolatesresistant to approved drugs. These viruses were obtained from the NIHAIDS Research and Reference Reagent Program. Assays were performed usingvirus propagated in PBMCs with a p24 endpoint (above), or using cellline targets (MT-2 cells) and a cell killing endpoint. The MT-2 assayformat was as follows. Serial dilutions of DSB, or each approved drug,were prepared in 96 well plates. To each sample well was added mediacontaining MT-2 cells at 3×10⁵ cells/mL and virus inoculum at aconcentration necessary to result in 80% killing of the cell targets at5 days post-infection (PI). On day 5 post-infection, virus-induced cellkilling was determined by the XTT method and the inhibitory activity ofthe compound was determined.

Table 6 shows the potent anti-viral activity of DSB against a panel ofdrug-resistant HIV-1 isolates. The results were not significantlydifferent from those obtained with the panel of wild-type isolates(Table 5), demonstrating that DSB retains its activity against virusstrains resistant to all of the major classes of approved drugs.

TABLE 6 Inhibitory activity (nM IC₅₀) of DSB against a panel of drugresistant HIV-1 isolates. Assays were done in fresh PBMC with a p24endpoint except for the NNRTI-resistant isolates that were performed inMT-2 cells with a cell viability (XTT) endpoint. Virus Co- IsolateReceptor IC₅₀(nM) # Mutation(s) usage DSB AZT Nevirapine IndinavirNRTI-resistant 1 K70R R5/X4 4.4 86.4 (54X)* ND  9.8 T215Y/F 2 K70R R5/X44.2 63.4 (40X)  ND  6.1 T215Y/F NNRTI-resistant 3 Y181C X4 1.0 5.1 >3800 2.5 (>177X) 4 K103N X4 1.3 2.0  2630  4.5 Y181C  (122X)Protease-resistant 5 V82A X4 5.6 13.1  ND 39.7 (12X) 6 I84V X4 5.5 14.4 ND 32.7 (10X) 7 L10R/M46I/ X4 12.9 3.5 ND 72.5 L63P/V82T/I84V (23X)*Fold Resistance. Note: R5 and X4 refer to the chemokine receptors CCR5and CXCR4 respectively.

Example 3 DSB Inhibits HIV-1 Replication at a Late Step in the VirusLife Cycle

To distinguish the inhibitory activity of DSB against early and latereplication targets, a multinuclear activation of a galactosidaseindicator (MAGI) assay was used. In this assay, the targets are HeLacells stably expressing CD4, CXCR4, CCR5 and a reporter constructconsisting of the -galactosidase gene (modified to localize to thenucleus) driven by a truncated HIV-1 LTR. Infection of these cellsresults in expression of Tat that drives activation of theβ-galactosidase reporter gene. Expression of β-galactosidase in infectedcells is detected using the chromogenic substrate X-gal. As shown inTable 7, the entry inhibitor T-20, the NRTI AZT and the NNRTI nevirapinecaused significant reductions in β-galactosidase gene expression inHIV-1 infected MAGI cells due to their ability to disrupt early steps inviral replication that affect Tat protein expression. In contrast, theprotease inhibitor indinavir targets a late step in virus replication(following Tat expression) and does not prevent β-galactosidase geneexpression in this system. Similar results were obtained with DSB aswith indinavir, indicating that DSB blocks virus replication at a timepoint following the completion of proviral DNA integration and synthesisof the viral transactivating protein (Table 7).

TABLE 7 Effect of DSB and inhibitors of entry (the gp41 peptide T-20),RT (AZT and Nevirapine) and protease (indinavir) on expression ofb-galactosidase in HIV-1 infected MAGI cells. The DMSO control containedno drug. Inhibitor DMSO T-20 AZT Nevirapine Indinavir DSB % Decrease 098 82 85 10 12 (β- galactosidase expression)

Kanamoto et al. (Antimicrob. Agents Chemother., April; 45(4):1225-30,(2002)) have also reported that DSB acts at a late step in HIVreplication. However, they reported that the compound inhibits releaseof virus from chronically-infected cells. In contrast, our data using avariety of experimental systems indicate that DSB does not have asignificant effect on virus release (e.g. Example 6).

Example 4 DSB does not Inhibit HIV-1 Protease Activity

It was previously determined that DSB had no effect on HIV-1 proteasefunction using a cell-free fluorometric assay that characterized enzymeactivity by following the cleavage of a synthetic peptide substrate. Theresults of these experiments indicated that at concentrations up to 50μg/mL that DSB had no effect on protease function. As a result of theobservation that DSB blocks virus replication at a late step, studieswere also performed using a recombinant form of the Gag protein, whichis a more relevant system than the synthetic peptide substrate used inthe initial assays. The use of the recombinant Gag protein as substrateresulted in a similar experimental outcome. In these experiments DSB didnot disrupt protease-mediated Gag protein processing at concentrationsas high as 50 μg/mL. In contrast, as expected, the protease inhibitorindinavir blocked Gag protein processing at 5 μg/mL (FIG. 1).

Example 5 DSB Causes a Defect in the Final Step of Gag Processing(CA-SP1 Cleavage) that has been Associated with Viral Maturation Defects

In order to better define DSB's mechanism of action, a detailedexamination was undertaken of the virus produced from HIV-1-infectedcell lines treated with DSB. Briefly, H9 cells chronically infected withthe HIV-1_(IIIB) isolate were treated with DSB at 1 μg/mL for a periodof 48 hrs. Indinavir was used as a control. At the 48 hr time-point,spent media was removed and fresh media containing compound was added.At 24, 48 and 72 hrs post fresh compound addition, both cells andsupernatant were recovered for analysis. The level of virus in theculture supernatant was determined and western blots were used tocharacterize viral protein production in both cell-associated andcell-free virus. As observed in previous experiments, DSB did not causea significant reduction in the amount of virus produced by chronicallyinfected H9 cells, however, there was a defect in Gag processing in bothcell-associated and cell-free virus. This defect took the form of anadditional band in the western blots corresponding to p25 (FIG. 2). Thisp25 band results from the incomplete processing of the capsid CA-SP1precursor. DSB treatment of HIV-2 and SIV chronically infected celllines exhibited normal Gag processing consistent with the observed lackof antiviral activity against these viruses. The Gag processing defectseen in the presence of DSB is completely distinct from that observedwith the protease inhibitor indinavir (FIG. 2). As discussed above,mutations at the p25 to p24 cleavage site that prevent processing areassociated with defects in viral maturation and infectivity (Wiegers K.et al., J. Virol. 72:2846-54 (1998)).

As previously discussed (C. T. Wild et al., XIV Int. AIDS Conf.Barcelona, Spain, Abstract MoPeA3030, (July 2002)), abnormal p25 to p24processing is also seen in other maturation budding defects. Theseinclude mutations in the Gag late domain (PTAP) or defects in TSG-101mediated viral assembly that disrupt budding (Garrus, J. E et al., Cell,107:55-65, (2001); Demirov, D. G. et al., J. Virology 76:105-117,(2002)). However, these mutations cause inhibition of virus release,while DSB treatment does not have a significant effect on virus release.The morphology of these maturation/budding mutants is also quitedistinct from that following DSB-treatment (see Example 6).

In addition, mutations that interfere with viral RNA dimerization andlead to the production of immature virus with defective core structuresgive a similar Gag processing phenotype (Liang, C. et al., J. Virology,73:6147-6151, (1999)). However, in those cases RNA incorporation isinhibited and the morphology of particles released is distinct fromthose following DSB treatment (see Example 6).

Example 6 DSB Treatment Effects HIV-1 Maturation as Determined byElectron Microscopy (EM)

It has been demonstrated that mutations in HIV-1 Gag that disrupt p25 top24 processing give rise to non-infectious viral particles characterizedby an internal morphology distinct from normal virus (Wiegers K. et al.,J. Virol. 72:2846-54 (1998)). To determine if virus generated in thepresence of DSB exhibited this distinct morphology the followingexperiment was carried out.

HeLa cells were transfected with HIV-1 infectious molecular clone pNL4-3and treated as described previously with DSB. Following treatment,DSB-treated infected cells were fixed in glutaraldehyde and analyzed byEM. The results of this analysis are shown in FIG. 3.

These results are consistent with a compound that disrupts p25 to p24processing which generates non-infectious morphologically aberrant viralparticles.

3-O-(3′,3′-dimethylsuccinyl)betulinic acid (DSB) is an example of acompound that disrupts p25 to p24 processing and potently inhibits HIV-1replication. However, this compound does not inhibit PR activity, andits action is specific for the p25 to p24 processing step, not othersteps in Gag processing. Furthermore, DSB treatment results in theaberrant HIV particle morphology described above.

Example 7

In vitro selection for HIV-1 isolates resistant to compounds thatdisrupt the processing of the viral Gag capsid (CA) protein from theCA-spacer peptide 1 protein precursor.

A series of experiments were performed to select for viruses resistantto inhibition by 3-O-(3′,3′-dimethylsuccinyl)betulinic acid (DSB), aninhibitor HIV-1 maturation. For each experiment, either NL4-3 or RFvirus isolate was used to infect two cell cultures. Following infection,one culture was maintained in growth medium containing DSB, while theother culture was maintained in parallel in growth medium lacking DSB.

In one experiment, H9 cells that had been infected with RF virus weremaintained in the presence or absence of increasing concentrations ofDSB (0.05-1.6 μg/ml). The cells were passaged every 2-3 days with theaddition of fresh drug. Virus replication was monitored by p24 ELISAevery 7 days. At that time, DSB-treated cultures with high levels of p24were passaged by co-cultivation with fresh uninfected H9 cells at a 1:1ratio of cells in the presence of 1× or 2× the original concentration ofDSB. After 8 weeks of co-cultivation, cell-free virus was collected fromthe culture containing DSB at a concentration of 1.6 μg/ml and used toinfect fresh H9 cells. Every 7 days, virus from cultures containing highlevels of p24 was passaged by cell-free infection in the presence of 1×or 2× the original concentration of DSB. After 5 weeks of cell-freepassaging, virus from the culture containing 3.2 μg/ml DSB was collectedand used to infect MT-2 cells. Virus replication in the MT-2 cells, wasmonitored by observing syncytia formation microscopically. Every 1-3days, the cells were washed to remove input virus, and fresh drug wasadded to the culture under selection. Every 3-4 days, following theemergence of extensive syncytia in the culture under selection,supernatant from each culture was collected and passed through a 0.45 μmfilter to remove cell debris. This filtered virus supernatant was thenused to infect fresh MT-2 cells in the presence or absence of freshdrug. After 4 rounds of cell-free infection (approximately 2 weeks inculture), with the concentration of drug at 3.2 μg/ml, virus stocks werecollected and frozen for further analysis.

In a second experiment, a stock of virus derived from the molecularclone pNL4-3 (5.7×104 TCID50) was used to infect MT-2 cells (6×106cells) and cultures were maintained in the presence or absence ofPA-457DSB at a concentration of 1.6 μg/ml. Every 1-3 days, the cellswere washed to remove input virus, and fresh drug was added to theculture under selection. Virus replication was monitored by observingsyncytia formation microscopically. Every 3-7 days, following theemergence of extensive syncytia in the culture under selection,supernatant from each culture was collected and passed through a 0.45 μmfilter to remove cell debris. This filtered virus supernatant was thenused to infect fresh MT-2 cells in the presence or absence of freshdrug. After 5 rounds of cell-free infection, and every other roundthereafter, the concentration of drug was doubled. After 10 rounds ofcell-free infection (approximately 7 weeks in culture), when theconcentration of drug reached 12.8 μg/ml, virus stocks were collectedand frozen for further analysis.

Example 8

Characterization of HIV-1 isolates selected for resistance to compoundsthat disrupt the processing of the viral Gag capsid (CA) protein fromthe CA-spacer peptide 1 protein precursor.

Virus stocks derived as described above were further analyzed bothphenotypically and genotypically to characterize the nature of theirdrug-resistance. The resistance of the viruses to3-O-(3′,3′-dimethylsuccinyl)-betulinic acid (DSB) was determined invirus replication assays. Briefly, the virus stocks were first titeredin H9 cells by quantitating the levels of p24 (by ELISA) in cultures 8days after infection with serial 4-fold dilutions of virus. Virus inputwas then normalized for a second assay in which each virus is culturedfor 8 days in the presence of serial 4-fold dilutions of drug. The IC50for each virus was determined as the dilution of drug that reduced thep24 endpoint level by 50% as compared to the no-drug control. In theseassays, the two independently derived virus stocks resulted in IC₅₀values greater than 2 μM for DSB, as compared to an IC₅₀ of 0.02 μM forvirus that had been cultured in parallel in the absence of drug. In asubsequent series of experiments, the A364V mutation was engineered intothe HIV-1 NL4-3 proviral DNA, which was subsequently transfected intoHeLa cells. Resulting virus was collected and used to test the activityof DSB in a viral replication assay, as described above. In theseassays, the DSB-resistant virus resulted in an IC₅₀ value of 0.1 μMwhereas wild-type NL4-3 gave an IC₅₀ value of 0.01 μM.

To determine if the resistant viruses were able to escape the CA-SP1cleavage defect caused by DSB in wild-type virus, stocks of each virusgrown in either the presence or absence of drug were analyzed by Westernblot. Virus was pelleted through a 20% sucrose cushion from filteredculture supernatants that were collected 60 hr post-infection and 18 hrafter the cells had been washed and fresh drug added. The viruses werelysed, and the amount of each virus was normalized by quantitating p24levels in each sample. Western blot analysis of the viral proteins ineach sample demonstrated that the drug-resistant viruses did not containthe CA-SP1 product in the presence of DSB, confirming that these viruseswere resistant to the effects of the drug on this cleavage event.

Finally, to identify the genetic determinants of DSB resistance, theentire Gag and PR coding regions of the viral genomes were amplified byhigh-fidelity RT-PCR for sequencing. The viral RNA was purified fromeach virus lysate prepared as described above and digested with DNase toremove any contaminating DNA. The RT-PCR products were then gel-purifiedto remove any non-specific PCR products. Finally, both strands of theresulting DNA fragments were sequenced using overlapping a series ofprimers. Two amino acid mutations were identified that are independentlycapable of conferring resistance to DSB, an alanine to valinesubstitution in the Gag polyprotein at residue 364 in the NL4-3 isolateand at residue 366 in the RF isolate. These are the first and the thirdresidues, respectively, downstream of the CA-SP1 cleavage site (theN-terminus of SP1). Alanine is highly conserved at each of thesepositions throughout all HIV-1 clades in the database.

Having now fully described this invention, it will be understood tothose of ordinary skill in the art that the same can be performed withina wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or anyembodiment thereof. All patents, applications and publications citedherein are fully incorporated by reference in their entirety.

1. A method of determining if an individual is infected with HIV-1 thatis susceptible to inhibition by a compound that inhibits p25 (CA-SP1)processing to p24 (CA), comprising taking a sample of blood from thepatient, and determining whether HIV-1 gag in said sample is susceptibleto inhibition of p25 (CA-SP1) processing to p24 (CA) by the compound. 2.The method of claim 1, which comprises determining whether said HIV-1gag contains a mutation that results in a decrease in inhibition ofprocessing by said compound.
 3. The method of claim 2, which comprisesgenotyping said HIV-1 gag.
 4. The method of claim 3, which comprisesgenotyping the region of the CA-SP1 cleavage site.
 5. The method ofclaim 3, which comprises determining whether said HIV-1 gag encodes asubstitution of Ala to Val at a position corresponding to residue 364 ofHIV-1 Gag (residue 1 of SP1).
 6. The method of claim 3, which comprisesdetermining whether said HIV-1 gag encodes a substitution of Ala to Valat a position corresponding to residue 366 of HIV-1 Gag (residue 3 ofSP1).
 7. The method of claim 3, which comprises determining whether saidHIV-1 gag encodes a substitution of H is to Tyr at a positioncorresponding to residue 358 of HIV-1 Gag.
 8. The method of claim 3,which comprises determining whether said HIV-1 gag encodes asubstitution of Leu to Phe at a position corresponding to residue 363 ofHIV-1 Gag (the C-terminal residue of CA).
 9. The method of claim 3,which comprises determining whether said HIV-1 gag encodes asubstitution of Leu to Met at a position corresponding to residue 363 ofHIV-1 Gag (the C-terminal residue of CA).
 10. The method of claim 3,which comprises determining whether said HIV-1 gag encodes a deletion ofthe residue at a position corresponding to residue 370 of HIV-1 Gag(position 7 of SP1).
 11. The method of claim 3, which comprisesdetermining whether said HIV-1 gag encodes a substitution of Ala to Valat a position corresponding to residue 366 of HIV-1 Gag (position 3 ofSP1) and a Ser residue at a position corresponding to residue 357 ofHIV-1 Gag.
 12. The method of claim 3, which comprises determiningwhether said HIV-1 gag encodes a substitution of Ala to Val at aposition corresponding to residue 366 of HIV-1 Gag (position 3 of SP1)and a substitution of Gly to Ser at a position corresponding to residue357 of HIV-1 Gag.